CN105378486A - Automated diagnostic analyzers having vertically arranged carousels and related methods - Google Patents

Automated diagnostic analyzers having vertically arranged carousels and related methods Download PDF

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CN105378486A
CN105378486A CN201480033702.8A CN201480033702A CN105378486A CN 105378486 A CN105378486 A CN 105378486A CN 201480033702 A CN201480033702 A CN 201480033702A CN 105378486 A CN105378486 A CN 105378486A
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fatty acid
recombinant microorganism
hydroxy fatty
polypeptide
derivative
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A·W·席尔默
H·王
S·B·德尔卡黛莉
胡志浩
L·G·霍姆
B·朱
C·常
E·E·波波瓦
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Genomatica Inc
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LS9 Inc
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    • C12Y114/14—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen (1.14.14)
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    • C12Y114/15003—Alkane 1-monooxygenase (1.14.15.3)
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Abstract

Example automated diagnostic analyzers and methods for using the same are disclosed herein. An example apparatus described herein includes a first carousel rotatably coupled to a base and having a first axis of rotation. The example apparatus includes a second carousel rotatably coupled to the base and vertically spaced over the first carousel such that at least a portion of the second carousel is disposed over the first carousel. In the example apparatus, the second carousel has a second axis of rotation and a plurality of vessels. The example apparatus also includes a pipetting mechanism offset from the second axis of rotation. The example pipetting mechanism is to access the first carousel and the second carousel.

Description

Produce the method for the hydroxylated derivative of fatty acid of OMEGA-
The cross reference of related application
This application claims the U.S. Provisional Application No.61/835 submitted on June 14th, 2013, the rights and interests of 464, whole disclosures of this application are incorporated herein by reference.
Sequence table
The application comprises sequence table, and this sequence table is electronically submitted to ASCII fromat, and is incorporated in full herein with way of reference.ASCII described in creating on June 16th, 2014 copies called after LS00048PCT_SL.txt, and size is 342,103 bytes.
Technical field
The present invention openly relates to the hydroxylated derivative of fatty acid of omega-and produces their method.In the present invention, the present invention openly covers and provides the new of the hydroxylated derivative of fatty acid of omega-and eco-friendly production method with high-purity and high yield.In addition, the present invention openly covers the recombinant microorganism organism being produced the hydroxylated derivative of fatty acid of omega-by selective fermentation.
Background technology
Omega-hydroxylated (ω-hydroxyl) derivative of fatty acid has many commercial uses as the composition of industrial reagent.Industry member has confirmed polytype ω-hydroxy fatty acid derivative, comprises ω-hydroxy fatty acid; ω-hydroxy fatty acid methyl ester; ω-oxo fatty acid; Omega-amino-fatty acid; Omega-amino-fatty acid methyl ester; Alpha-, omega-diacid (α, ω-diacid); Omega-carboxylic fatty acids methyl ester (ω-carboxylic fatty acids methyl ester); Alpha-, omega-diester (α, ω-diester); Alpha-, omega-glycol (α, omega-diol) etc.These molecules are also important as the precursor of other compounds multiple.Such as α, alpha, omega-dicarboxylic acid and other α, ω-bifunctioanl molecule is important chemicals in the commercial Application of fluoropolymer resin, working metal liquid, bonding agent, corrosion inhibitor, capacitor electrolyte, diester syntholube, fiber, powder coating medicine, plastifier, polyester coating, epoxy resin, polyamide, spices (flavor), essence (fragrance), surfactant, washing agent, adjuvant etc.At present, ω-hydroxy fatty acid derivative is still primarily of petroleum based material or transformed by the biology of paraffin and fatty acid and obtain.Chemical method for the production of these compounds needs to use hazardous agents, and is energy-intensive and environmental cost is large.On the contrary, although emerging fermentation approach is considered to friendly process, is still very expensive and is confined to the product of the several types that can prepare.Therefore, the technique of directly being produced polytype and functional ω-hydroxy fatty acid derivative by reproducible feed is not only safer to environment, but also has more cost benefit significantly.The present invention openly solves this needs.
Summary of the invention
An aspect disclosed by the invention provides under the carbon source obtained by reproducible feed exists, when growing in fermentation liquor, for producing the recombinant microorganism organism of omega-hydroxylated (ω-hydroxyl) derivative of fatty acid in vivo.Described microbial organisms comprises the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the thioesterase of EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase of EC1.14.15.3.Described modified ω-hydroxylase has modified cytochrome P 450 monooxygenases (P450) enzymatic activity, and the ω-position of catalytic hydrocarbon chain effectively in vivo.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, described CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.In the present invention, described ω-hydroxylase hybrid fusion protein qualitative change body has at least one sudden change at amino acid position 27,82,141,178,231,309,407,415,516,666 and/or 796.Described recombinant microorganism organism produces ω-hydroxy fatty acid derivative, includes but not limited to ω-hydroxy fatty acid and ω-hydroxy fatty acid methyl ester.
Disclosed by the inventionly another aspect provides under the carbon source that obtained by reproducible feed exists, when growing in fermentation liquor, for producing the recombinant microorganism organism of ω-hydroxy fatty acid derivative in vivo.Described microbial organisms comprises the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the thioesterase of EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase of EC1.14.15.3.ω-the hydroxylase of described modification has modified cytochrome P 450 monooxygenases (P450) enzymatic activity, and the ω-position of catalytic hydrocarbon chain effectively in vivo.In an aspect, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.1.1/2, or the alcohol oxidase of EC1.1.3.13 or EC1.1.3.20.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.Described recombinant microorganism organism produces ω-hydroxy fatty acid derivative, includes but not limited to ω-oxo fatty acid and ω-oxo fatty acid methyl ester.
Disclosed by the inventionly another aspect provides under the carbon source that obtained by reproducible feed exists, when growing in fermentation liquor, for producing the recombinant microorganism organism of ω-hydroxy fatty acid derivative in vivo.Described microbial organisms comprises the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the thioesterase of EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase (cytochrome P 450 monooxygenases) of EC1.14.15.3.In an aspect, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.1.1/2, or the alcohol oxidase of EC1.1.3.13 or EC1.1.3.20.In one aspect of the method, modified microbial organisms expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the aldehyde dehydrogenase of EC1.2.1.3/4/5 or the aldehyde oxidase of EC1.2.3.1.ω-the hydroxylase of described modification has modified cytochrome P 450 monooxygenases (P450) enzymatic activity, and the ω-position of catalytic hydrocarbon chain effectively in vivo.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.Described recombinant microorganism organism produces ω-hydroxy fatty acid derivative, and include but not limited to ω-hydroxy fatty acid derivative, it is α, ω-diacid or ω-carboxylic fatty acids methyl ester.
And another aspect of the present invention provides under the carbon source obtained by reproducible feed exists, when growing in fermentation liquor, for producing the recombinant microorganism organism of ω-hydroxy fatty acid derivative in vivo.Described microbial organisms comprises the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises EC3.1.2.-, the thioesterase of 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase of EC1.14.15.3.In an aspect, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.1.1/2, or the alcohol oxidase of EC1.1.3.13 or EC1.1.3.20.In one aspect of the method, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the aldehyde dehydrogenase of EC1.2.1.3/4/5 or the aldehyde oxidase of EC1.2.3.1.In one aspect of the method, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, and described polypeptide comprises the acyl group-CoA ligase of EC6.2.1.3 or the acyl group-CoA transferase of EC2.8.3.6.ω-the hydroxylase of described modification has modified cytochrome P 450 monooxygenases (P450) enzymatic activity, and the ω-position of catalytic hydrocarbon chain effectively in vivo.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.Described recombinant microorganism organism produces ω-hydroxy fatty acid derivative, and include but not limited to ω-hydroxy fatty acid derivative, it is α, ω-diester.
The present invention openly covers further under the carbon source obtained by reproducible feed exists, when growing in fermentation liquor, for producing the recombinant microorganism organism of ω-hydroxy fatty acid derivative in vivo.Described microbial organisms comprises the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises EC3.1.2.-, the thioesterase of 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase of EC1.14.15.3.In one aspect of the method, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.1.1/2 or the alcohol oxidase of EC1.1.3.13 or EC1.1.3.20.In one aspect of the method, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the aminopherase of EC2.6.1 or the amine dehydrogenase of EC1.4.9, EC1.4.98 or EC1.4.99.Described ω-hydroxylase has modified cytochrome P 450 monooxygenases (P450) enzymatic activity, and the ω-position of catalytic hydrocarbon chain effectively in vivo.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.Described recombinant microorganism organism produces ω-hydroxy fatty acid derivative, and it includes but not limited to omega-amino-fatty acid and omega-amino-fatty acid methyl ester.
Disclosed by the inventionly another aspect provides under the carbon source that obtained by reproducible feed exists, when growing in fermentation liquor, for producing the recombinant microorganism organism of ω-hydroxy fatty acid derivative in vivo.Described microbial organisms comprises the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the thioesterase of EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase of EC1.14.15.3.In an aspect, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.-.-or the carboxylate reductase of 1.2.99.Described ω-hydroxylase has modified cytochrome P 450 monooxygenases (P450) enzymatic activity, and the ω-position of catalytic hydrocarbon chain effectively in vivo.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.Described recombinant microorganism organism produces ω-hydroxy fatty acid derivative, and it includes but not limited to ω-hydroxy fatty acid derivative, and it is α, omega-diol.
The present invention openly further contemplates the cell culture comprising microbial organisms (as mentioned above), wherein said cell culture produces ω-hydroxy fatty acid derivative, include but not limited to ω-hydroxy fatty acid, comprise ω-hydroxyl free fatty acid; ω-hydroxy fatty acid methyl ester; ω-oxo fatty acid; ω-oxo fatty acid methyl ester; α, ω-diacid; α, omega-diol; α, ω-diester; ω-carboxylic fatty acids methyl ester; Omega-amino-fatty acid; With omega-amino-fatty acid methyl ester.
The method that another aspect provides production ω-hydroxy fatty acid derivative disclosed by the invention, it is included in fermentation liquor provides recombinant microorganism organism, described microbial organisms has the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the thioesterase of EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase of EC1.14.15.3.Described method comprises further and being added in fermentation liquor by the reproducible feed comprising carbon source, and by separation of fermentative broth ω-hydroxy fatty acid derivative.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.In an aspect, the ω-hydroxy fatty acid derivative produced is ω-hydroxyl free fatty acid or ω-hydroxy fatty acid methyl ester.
The method that another aspect provides production ω-hydroxy fatty acid derivative disclosed by the invention, it is included in fermentation liquor provides recombinant microorganism organism, described microbial organisms has the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the thioesterase of EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the ω-hydroxylase (cytochrome P 450 monooxygenases) of EC1.14.15.3.In in concrete at one, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.1.1/2 or the alcohol oxidase of EC1.1.3.13 or EC1.1.3.20.Described method comprises further and being added in fermentation liquor by the reproducible feed comprising carbon source, and by separation of fermentative broth ω-hydroxy fatty acid derivative.In an aspect, the ω-hydroxy fatty acid derivative produced is ω-oxo fatty acid or ω-oxo fatty acid methyl ester.
The method that another aspect provides production ω-hydroxy fatty acid derivative disclosed by the invention, it is included in fermentation liquor provides recombinant microorganism organism, described microbial organisms has the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the thioesterase of EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase of EC1.14.15.3.In in concrete at one, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.1.1/2 or the alcohol oxidase of EC1.1.3.13 or EC1.1.3.20.In in another is specific, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the aldehyde dehydrogenase of EC1.2.1.3/4/5 or the aldehyde oxidase of EC1.2.3.1.Described method comprises further and being added in fermentation liquor by the reproducible feed comprising carbon source, and by separation of fermentative broth ω-hydroxy fatty acid derivative.In an aspect, the ω-hydroxy fatty acid derivative produced is α, ω-diacid or α, omega-3-fatty acids dimethyl esters.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.
The method that another aspect provides production ω-hydroxy fatty acid derivative disclosed by the invention, it is included in fermentation liquor provides recombinant microorganism organism, described microbial organisms has the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the thioesterase of EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase of EC1.14.15.3.In in concrete at one, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.1.1/2 or the alcohol oxidase of EC1.1.3.13 or EC1.1.3.20.In in another is specific, described recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the aminopherase of EC2.6.1 or the amine dehydrogenase of EC1.4.9, EC1.4.98 or EC1.4.99.Described method comprises further and being added in fermentation liquor by the reproducible feed comprising carbon source, and by separation of fermentative broth ω-hydroxy fatty acid derivative.In an aspect, the ω-hydroxy fatty acid derivative produced is omega-amino-fatty acid or omega-amino-fatty acid methyl ester.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.
The present invention openly further contemplates the method for producing ω-hydroxy fatty acid derivative, it is included in fermentation liquor provides recombinant microorganism organism, described microbial organisms has the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the thioesterase of EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase of EC1.14.15.3.In in concrete at one, described recombinant microorganism organism expresses the nucleic acid of following polypeptide of encoding further through transformation, described polypeptide comprises the carboxylate reductase of EC1.2.99.6 and/or the alcohol dehydrogenase of EC1.1.-.-.Described method comprises further and being added in fermentation liquor by the reproducible feed comprising carbon source, and by separation of fermentative broth ω-hydroxy fatty acid derivative.In an aspect, the ω-hydroxy fatty acid derivative produced is α, omega-diol.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.
Disclosed by the inventionly another aspect provides method (as mentioned above), wherein reproducible feed is carbon back, and it includes but not limited to corn, sugarcane, Chinese sorghum, beet, switchgrass, fresh stover, straw, timber, paper pulp, sewage, rubbish, cellulose municipal waste, waste gas, synthetic gas and carbon dioxide.In an aspect, carbon source is selected from glucose, fructose, mannose, galactose, wood sugar, arabinose, FOS, galactooligosaccharide, starch, cellulose, colloid, xylan, sucrose, maltose, cellobiose, turanose, hemicellulose, methylcellulose, sodium carboxymethyl cellulose, succinate, lactate, acetic acid esters, ethanol, methyl alcohol, glycerine and their potpourri.
The present invention openly further contemplates the polymer composition produced by the method disclosed in the present (as mentioned above), and wherein said polymer composition is including but not limited to polyurethane, polyester polyol, vibrin, alkyl coating resin, glass fiber resin, gel coat resin and thermoplastic polyester (polyesterthermoplastic).
Disclosed by the inventionly another aspect provides under the carbon source that obtained by reproducible feed exists, when growing in fermentation liquor, for producing the recombinant microorganism organism of ω-hydroxy fatty acid derivative in vivo, described microbial organisms comprises the approach of at least 3 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the acyl-acp reductase of EC1.2.1.42; The alcohol dehydrogenase of EC1.1.-.-; And the modified ω-hydroxylase of EC1.14.15.3.Described ω-hydroxylase has modified cytochrome P 450 monooxygenases (P450) enzymatic activity, and the ω-position of catalytic hydrocarbon chain effectively in vivo.In one embodiment, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.ω-the hydroxy fatty acid derivative produced is α, omega-diol.In an aspect, additionally provide cell culture, it comprises microbial organisms disclosed in this invention (as mentioned above).
But, the method that another aspect provides production ω-hydroxy fatty acid derivative disclosed by the invention, it is included in fermentation liquor provides recombinant microorganism organism, described microbial organisms comprises the approach of at least 3 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the acyl-acp reductase of EC1.2.1.42; The alcohol dehydrogenase of EC1.1.-.-; And the modified ω-hydroxylase of EC1.14.15.3.Described method comprises further and being added in fermentation liquor by the reproducible feed comprising carbon source, and by separation of fermentative broth ω-hydroxy fatty acid derivative.In an aspect, modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.In another embodiment, modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.In another embodiment, CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.In another embodiment, for the modified ω-hydroxylase of ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and there is one or more sudden change, comprise V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and/or A796V.In the present invention, ω-hydroxy fatty acid derivative is α, omega-diol.In an aspect, reproducible feed is carbon back, and comprises corn, sugarcane, Chinese sorghum, beet, switchgrass, fresh stover, straw, timber, paper pulp, sewage, rubbish, cellulose municipal waste, waste gas, synthetic gas and carbon dioxide.In yet another aspect, carbon source is selected from glucose, fructose, mannose, galactose, wood sugar, arabinose, FOS, galactooligosaccharide, starch, cellulose, colloid, xylan, sucrose, maltose, cellobiose, turanose, hemicellulose, methylcellulose, sodium carboxymethyl cellulose, succinate, lactate, acetic acid esters, ethanol, methyl alcohol, glycerine and their potpourri.The polymer composition produced by described method is also comprised in the present invention further, and wherein said polymer composition includes but not limited to polyurethane, polyester polyol, vibrin, alkyl coating resin, glass fiber resin, gel coat resin and thermoplastic polyester.
The present invention openly further contemplates the Flavor Chemicals composition produced by methods described herein (as mentioned above), and wherein said Flavor Chemicals composition is the saturated or unsaturated macrolide of C9 to C16.Described Flavor Chemicals composition comprises and is selected from Moschus lactones (ambrettolide), dihydro Moschus lactones, the macrolide of 15-hydroxy-pentadecanoic acid, the macrolide of 15-hydroxyl 15 carbon diluted acid and/or the chemical entities in other.
Accompanying drawing is sketched
When combine accompanying drawing read together the present invention open time, can understand better, wherein said accompanying drawing plays the effect of preferred embodiment illustrated.In addition, it should be understood that the present invention is openly not limited to specific embodiment disclosed in accompanying drawing.
Fig. 1 depicts the approach for the preparation of ω-hydroxy-carboxylic acid, ω-oxo-carboxylic acid, omega-amino--carboxylic acid and α, ω-diacid.The derivative of fatty acid with 12 carbon atoms is described as an example.
Fig. 2 A depicts the approach being prepared ω-hydroxy-carboxylic acid, omega-amino--carboxylic acid and α, ω-diacid by methyl ester intermediate.The derivative of fatty acid with 12 carbon atoms is described as an example.
Fig. 2 B depicts and prepares α by dimethyl esters intermediate, the approach of ω-diacid.The derivative of fatty acid with 12 carbon atoms is described as an example.
Fig. 3 depicts and uses thioesterase and carboxylate reductase to prepare α, the approach of omega-diol.The derivative of fatty acid with 12 carbon atoms is described as an example.
Fig. 4 depicts and uses acyl-acp reductase to prepare α, the approach of omega-diol.The derivative of fatty acid with 12 carbon atoms is described as an example.
Fig. 5 shows approach omega-amino-Carboxylic Acid being become lactams.The derivative of fatty acid with 12 carbon atoms is described as an example.
Fig. 6 shows the GC/MS chromatogram of the extract obtained by the recombinant escherichia coli strain of expressing cyp153A operon, and described bacterial strain can form sabinic acid when feeding dodecylic acid.In addition, also show the GC/MS chromatogram of the extract obtained by control strain (MG1655 Δ fadD).
Fig. 7 A to 7C shows the mass spectrum of the sabinic acid (reaching peak value 11.393 minutes time) (Fig. 7 C) of derivative sabinic acid (reaching peak value 12.297 minutes time) (Fig. 7 A), derivative sabinic acid true standard product (Fig. 7 B) and non-derived.Derivatization reagent is BSTFA+1%TMCS.Mass spectrum in Fig. 7 A and 7C derives from the extract of coli strain sAS.321.
Fig. 8 A to 8B shows the ion fragmentation mode of the sabinic acid (Fig. 8 B) of derivative sabinic acid (12-trimethylsiloxy dodecylic acid trimethyl silyl ester) (Fig. 8 A) and non-derived.
Fig. 9 shows the GC/MS chromatogram of the extract obtained by the recombinant escherichia coli strain of expressing CYP153A operon, and described bacterial strain can form sabinic acid when feeding dodecylic acid.Bacterial strain sAS.335 have expressed the CYP153A operon of improvement.
Figure 10 A to 10C shows the GC/MS chromatogram of the extract obtained by the coli strain of expressing CYP153A-RedRhF fused protein, described bacterial strain forms sabinic acid (Figure 10 A) by dodecylic acid, sabinic acid methyl ester (Figure 10 B) is formed by dodecylic acid methyl ester, and form 1,12-dodecanediol (Figure 10 C) by dodecanol.In addition, also show the extract of control strain MG1655 Δ fadD.
Figure 11 A to 11B depicts obtained by the extract of bacterial strain sAS.336 derive 1,12-dodecanediol (reaching peak value 11.948 minutes time) (Figure 11 A), and the mass spectrum of real derivative 1,12-dodecanediol standard items (Figure 11 B).Derivatization reagent is BSTFA+1%TMCS.
Figure 12 A to 12B depicts the mass spectrum of the sabinic acid methyl ester obtained by the extract of bacterial strain sAS.336.That illustrated therein is non-derived (reaching peak value 11.107 minutes time) (Figure 12 A) and derive (reaching peak value 11.662 minutes time) (Figure 12 B) sample.Derivatization reagent is BSTFA+1%TMCS.
Figure 13 A to 13B shows the ion fragmentation mode of 1,12-dodecanediol (Figure 13 A) without derivatization and sabinic acid methyl ester (Figure 13 B).
Figure 14 A to 14C shows the GC/MS chromatogram of the extract obtained by the recombinant escherichia coli strain of expressing CYP153A-RedRhF fused protein, described bacterial strain produces ω-OH fatty acid (Figure 14 A), ω-OH fatty acid methyl ester (Figure 14 B) or α by glucose, omega-diol (Figure 14 C).All samples uses BSTFA+1%TMCS to carry out derivatization.
Figure 15 proves effectively to secrete ω-hydroxy fatty acid from production cell.
Figure 16 shows 2 kinds of coli strains produce ω-hydroxy fatty acid composition when grown on glucose.
Figure 17 shows the GC/MS chromatogram of the extract obtained by recombinant escherichia coli strain, and described bacterial strain expresses CYP153A-RedRhF fused protein, alcohol oxidase and aldehyde dehydrogenase, thus produces α, ω-diacid.All samples uses BSTFA+1%TMCS to carry out derivatization.
Figure 18 A to 18B depicts obtained by the extract of bacterial strain sEP.690 derive 1,14-tetracosandioic acid (reaching peak value 13.367 minutes time) (Figure 18 A), and the mass spectrum of real derivative 1,14-tetracosandioic acid standard items (Figure 18 B).Derivatization reagent is BSTFA+1%TMCS.
Figure 19 A shows the ion fragmentation mode of 1,14-derivative tetracosandioic acid.Derivatization reagent is BSTFA+1%TMCS.
Figure 20 shows the GC/MS chromatogram of the extract obtained by the recombinant escherichia coli strain of expressing cyp102A1 (F87A), and described bacterial strain produces a small amount of subterminal (such as ω-1, ω-2 and/or ω-3) hydroxylated fatty acid.In addition, also show the GC/MS chromatogram of the extract of control strain AlcV334.Described sample uses BSTFA+1%TMCS to carry out derivatization.
Figure 21 A to 21E depicts the mass spectrum at the peak of RT7.195 to 7.510 (deriving from Figure 20), and these peaks are identified as 11-hydroxy-dodecanoic acid (Figure 21 A), 10-hydroxy-dodecanoic acid (Figure 21 B), 9-hydroxy-dodecanoic acid (Figure 21 C), 8-hydroxy-dodecanoic acid (Figure 21 D), 7-hydroxy-dodecanoic acid (Figure 21 E).Describe to diagnose ion fragmentation for each hydroxylation position.Sample uses BSTFA+1%TMCS to carry out derivatization.
Figure 22 A to 22B shows the GC/MS chromatogram by the extract of the recombinant escherichia coli strain of expressing cyp102A7-protein, and described bacterial strain produces ω-1, ω-2 and ω-3 hydroxy fatty acid (Figure 22 A) or ω-1, ω-2 and ω-3 hydroxy fatty alcohols (Figure 22 B) by glucose.All samples uses BSTFA+1%TMCS to carry out derivatization.
Figure 23 A to 23C shows mass spectrum and the ion fragmentation mode of derivative 9-hydroxydodecanoic alcohol (reaching peak value 9.653 minutes time) (Figure 23 A), derivative 10-hydroxydodecanoic alcohol (reaching peak value 9.808 minutes time) (Figure 23 B) and derivative 11-hydroxydodecanoic alcohol (reaching peak value 9.905 minutes time) (Figure 23 C).Derivatization reagent is BSTFA+1%TMCS.Sample derives from the extract of coli strain XL963.
Figure 24 A to 24B shows proximal end (such as ω-1, ω-2 and/or ω-3) the hydroxylated fatty acid (Figure 24 A) of recombinant escherichia coli strain by glucose production and the amount of the hydroxylated fatty alcohol of proximal end (Figure 24 B) of expressing cyp102A7.
Figure 25 shows the ω-hydroxylated fatty acid produced by coli strain stEP.798.
Figure 26 shows the α produced by coli strain L1017, ω-diacid.
Figure 27 shows the result of the microbial organisms of culture expression CYP153A-reductase hybrid fusion protein qualitative change body.As shown, the variant with V141T (SEQID:46) creates the highest ω-hydroxy-16 carbon olefin(e) acid and tires, and by the highest conversion of hexadecene acid.
Detailed Description Of The Invention
Summary
To research and develop for the production of the new of ω-hydroxy fatty acid derivative and eco-friendly method means to have significant improvement to industry.Described method can make these compounds effectively produce from the simple carbon source derived by reproducible feed.Specifically, described method provides from reproducible material (carbohydrates such as obtained by corn, rattan or lignocellulose biomass), or waste product (such as glycerine, waste gas, synthetic gas), or ω-hydroxy fatty acid derivative produced by the organic material (such as living beings, rock gas or carbon dioxide) improved.
More specifically, present disclose provides novel restructuring microbial organisms, reproducible feed (such as carbohydrates) is changed into specific ω-hydroxy fatty acid derivative through transformation by this organism, and it comprises ω-hydroxy fatty acid; ω-hydroxy-fatty acid methyl ester; ω-carboxyl-fatty acid methyl ester; ω-oxo fatty acid, omega-amino-fatty acid, omega-amino-fatty acid methyl ester, α, ω-diacid; α, ω-diester; α, omega-diol etc.Like this, bifunctional molecule includes but not limited to ω-hydroxy fatty acid; α, ω-hydroxy fatty alcohols; α, ω-hydroxy-fatty acid methyl ester; α, ω-hydramine; α, ω-diacid; α, ω-difatty acid methyl ester, α, omega-diol etc.Described recombinant microorganism organism can carry out the effective zymotechnique of cost for the production of these compounds.The present invention is openly contained microbial fatty acid metabolic and its intermediate is changed into specific chemicals.
Advantage disclosed by the invention has a lot.Present disclosure provides simpler production method, that is, use simple sweat, but not multiple chemistry and/or the technique of living things catalysis, because described sweat creates less waste product, thus its more fast, the lower and more environmental friendliness of cost.Use reproducible feed (continuable starting material) and/or industrial wastes (such as glycerine) to add another cost benefit as source material, and protect environment.Present disclosure provides the selectivity manufacture of specific objective product (that is, comprising the composition of the chemical entities with selectivity chain length and chemical action).Obtain various chemical functionalities to may be used in the application of new target market.
Definition
As used herein, term " the hydroxylated derivative of fatty acid of omega-", " ω-hydroxylated derivative of fatty acid " and " ω-hydroxy fatty acid derivative " and " ω-hydroxy fatty acid derivative " and " ω-OH derivative of fatty acid " can exchange use in the present invention, and refer to and derive from fatty acid metabolism, and the chemical entities that there is in omega position at least one OH group or derived by the intermediate in omega position with at least one OH group.In the present invention, " omega position " refers to regard to its uncle functional group, at the terminal carbon of its contrary end place derivative of fatty acid.This type of ω-hydroxy fatty acid derivative includes but not limited to ω-hydroxy fatty acid; ω-hydroxy-fatty acid methyl ester; ω-carboxyl-fatty acid methyl ester; ω-oxo fatty acid; Omega-amino-fatty acid; Omega-amino-fatty acid methyl ester; And α, ω-diacid; α, ω-diester; And α, omega-diol.Term " ω-hydroxy fatty acid derivative " comprises " α, ω-dual functional derivative of fatty acid ".
" ω-hydroxy fatty acid derivative composition " is referred in the present invention and is produced by recombinant host cell, and usually comprise the potpourri (α of such as different chain length and/or saturation degree and/or Branching characteristics, the ω-diacid of the ω-hydroxy fatty acid derivative of the particular type with different chain length and/or saturation degree and/or Branching characteristics; Or the α of different chain length and/or saturation degree and/or Branching characteristics, ω-diester; Or the α of different chain length and/or saturation degree and/or Branching characteristics, omega-diol etc.).In some cases, ω-hydroxy fatty acid derivative composition comprises the ω-hydroxy fatty acid derivative of a most of type, such as 1,12-dodecene glycol, or 1,14-tetradecane diols, or juniperic acid methyl ester, or 16-hydroxyl-gaidic acid, or 15-hydroxyl-pentadecanoic acid, or 15-hydroxyl 15 carbon diluted acid, or 18-hydroxyl octadecenic acid, or any one methyl ester etc. of these derivative of fatty acid.In other cases, ω-hydroxy fatty acid derivative composition comprises the potpourri of the ω-hydroxy fatty acid derivative more than a type, to provide the composition of specific design (such as to have about 20%1 in identical composition, 12-dodecene glycol and about 80%1,16-hexadecane diol can as this type of examples).
Term " subterminal " hydroxylated derivative of fatty acid refers in omega-1 position, and/or omega-2 position, and/or omega-3 position, and/or (such as ω-1, ω-2 and/or ω-3 etc.) place such as omega-4 position has the chemical entities of at least one OH group (or obtaining by the intermediate with at least one OH group is derivative).Exemplary kind is ω-1, ω-2 and/or ω-3-hydroxy fatty acid; Or ω-1-hydroxy fatty acid methyl ester; Or ω-1, ω-2, ω-3, ω-4, and/or ω-5-hydroxy-dodecanoic acid etc.
Term " enzyme classification (EC) number " refers to the numbering representing certain enzyme activity.EC numbering under the naming system of enzyme, according to the reaction of their institute's catalysis by enzyme classification.EC numbering specifies enzymatic reaction.If the identical reaction of the different enzymatic that such as obtained by different organisms, then they have identical EC numbering.In addition, different protein folding things can the identical reaction of catalysis, will distribute identical EC numbering (such as nonhomologous bifunctional enzyme or NISE) thus.EC numbering is set up by the NK (IUBMB) of biological international chemistry and molecular biology federation, and its description can obtain on the website of the IUBMB enzyme naming system of internet.Such as cytochrome P 450 monooxygenases (P450) enzymatic activity (comprising ω-hydroxylase or omega oxidation enzyme enzymatic activity) is sorted under EC1.14.15.3 (also referred to as long acyl-[acyl group-Carrier-protein] reductase).Belong to the functional of the enzyme of P450 enzyme family is conservative at species to the most prokaryotes of next species.Therefore, different microbial species can have the identical enzymatic activity be categorized as under EC1.14.15.3.The enzymatic activity (as mentioned above) of the CYP153A-reductase hybrid fusion polypeptide that the example being characterized by the enzymatic activity of EC1.14.15.3 is discussed for the present invention or its variant.
Term " the modified ω-hydroxylase of EC1.14.15.3 " and " ω-hydroxylase of modification " can exchange use in the present invention, and refer to the cytochrome P 450 monooxygenases enzymatic activity of (such as in microbial organisms) in vivo ω position of catalytic hydrocarbon chain effectively.
Term " ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3 " and " ω-hydroxylase hybrid fusion protein qualitative change body " can exchange use in the present invention, and refer to the modified ω-hydroxylase hybrid fusion polypeptide in its amino acid sequence with at least one sudden change, thus in recombinant host cell, express ω-hydroxylase hybrid fusion protein qualitative change body, obtain thus compared with the expression of P450 fused protein natural in corresponding host cell, tire, productive rate and/or throughput rate obtain ω-OH fatty acid and/or the ω-OH fatty acid derived compositions of improvement.Such as when using ω-hydroxylase hybrid fusion protein qualitative change body to carry out transformant, this cell can express ω-hydroxylase hybrid fusion protein qualitative change body (such as recombinant cell).In one embodiment, by express ω-OH fatty acid that the cell of ω-hydroxylase hybrid fusion protein qualitative change body produces tire and/or productive rate is at least 2 times of the corresponding cell of expressing natural P450 fused protein.In another embodiment, by express ω-OH fatty acid or derivatives thereof that the cell of ω-hydroxylase hybrid fusion protein qualitative change body produces tire and/or productive rate is tiring of the corresponding cell of expressing natural P450 fused protein and/or productive rate at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times or at least about 10 times.In one embodiment, by express ω-OH fatty acid or derivatives thereof that the cell of ω-hydroxylase hybrid fusion protein qualitative change body produces tire and/or productive rate higher than express natural P450 fused protein corresponding cell tire and/or productive rate at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9% or at least about 10%.In another embodiment, make due to the expression of ω-hydroxylase hybrid fusion protein qualitative change body the ω-OH fatty acid or derivatives thereof produced in recombinant cell tire and/or productive rate higher than express natural P450 fused protein corresponding cell tire and/or productive rate at least about 20% at least about 80%.In some embodiments, ω-OH the fatty acid produced by described cell tire and/or productive rate higher than express natural P450 fused protein corresponding cell tire and/or productive rate at least approximately at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 100%.
Term " CYP153A-reductase hybrid fusion polypeptide " refers to the peptide sequence with SEQIDNO:6 with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.CYP153A-reductase hybrid fusion polypeptide is self-centered, and has the enzymatic activity that catalyze fatty acid reaction forms the ω-hydroxylase of ω-OH fatty acid.The example of CYP153A-reductase hybrid fusion polypeptide is the cyp153A-RedRhF type fused polypeptide of hybridization.
Term " accession number ", " NCBI accession number " or " GenBank accession number " refer to the numbering of the nucleotide sequence representing concrete.Sequence accession number discussed in this manual is obtained from the database that NCBI (NationalCenterforBiotechnologyInformation) provides, by theNationalInstitutesofHealth, U.S.A. safeguard, and the theUniProtKnowledgebase (UniProtKB) and Swiss-Prot database (also referred to as UniProtKB accession number) that are provided by theSwissInstituteofBioinformatics are provided.
As used herein, term " nucleotide " refers to the monomeric unit of polynucleotide, and it is made up of heterocyclic base, sugar and one or more phosphate.The base (guanine (G), adenine (A), cytimidine (C), thymine (T) and uracil (U)) of natural formation is generally the derivant of purine or pyrimidine, but it should be understood that the base analogue that natural and non-natural is formed also is included.The sugar of natural formation is pentose (pentose), ribodesose (it forms DNA) or ribose (it forms RNA), but it should be understood that the sugar analogue that natural and non-natural is formed also is included.Nucleic acid is connected by phosphate bond usually, thus forms nucleic acid or polynucleotide, but other keys (such as phosphorothioate linkages, boranophosphate key etc.) many known in the art.
As used herein, term " polynucleotide " refers to the polymkeric substance of nucleotide (RNA) and deoxynucleotide (DNA), and it can be strand or double-strand, and it can comprise the nucleotide of non-natural or change.Term " polynucleotide ", " nucleotide sequence " and " nucleotide sequence " can exchange use in the present invention, and it refers to the polymerized form of the nucleotide (RNA or DNA) of any length.These terms refer to the primary structure of molecule, therefore comprise double-strand and single stranded DNA and double-strand and single stranded RNA.This term comprises the analog of RNA or DNA formed by nucleotide analog and modified polynucleotide (such as but not limited to methylated and/or add the polynucleotide of cap) as equivalent.Polynucleotide can be any form, include but not limited to plasmid, virus, chromosome, EST, cDNA, mRNA and rRNA.
As used herein, term " polypeptide " and " protein " can exchange use, and it refers to the polymkeric substance of amino acid residue.Term " recombinant polypeptide " refers to the polypeptide produced by recombinant technique, wherein usually, is inserted in suitable expression vector, this carrier and then transformed host cell, thus produces polypeptide by expressed for coding protein DNA or RNA.
As used herein, term " homology " and " homology " refer to the polynucleotide or polypeptide that comprise following sequence, and wherein said sequence and corresponding polynucleotide or peptide sequence have the consistance of at least about 50%.Preferably, the polynucleotide of homology or polypeptide have following polynucleotide sequence or amino acid sequence, and this polynucleotide sequence or amino acid sequence and corresponding amino acid sequence or nucleotide sequence have at least about homology of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99%.As used herein, term sequence " homology " and sequence " consistance " can exchange use.Arbitrary those of ordinary skill of this area knows the method for the homology measured between two or more sequence well.In brief, " homology " calculated between two sequences can be carried out in the following manner.In order to reach the object that the best compares, sequence is compared (such as can introduce room to reach the object of best comparison in one or both of the first and second amino acid or nucleotide sequence, and nonhomologous sequence can be ignored compare object to reach).In preferred embodiments, for compare object, the length of the First ray of institute's comparison is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, even more preferably at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% of the length of the second sequence.Then, compare at the corresponding amino acid position of the first and second sequences or the amino acid residue at nucleotide position place or nucleotide.When the same amino acid residue of corresponding position during the position of First ray is by the second sequence or nucleotide occupy, then molecule is identical in this position.Percent homology between two sequences is the function of the quantity of the common same position of sequence, that takes into account the quantity in room and the length in each room, must introduce the quantity in room and the length in each room to reach the object of the best comparison of two sequences.The determination of the percent homology between the comparison of sequence and two sequences can use mathematical algorithm, such as BLAST (Altschuletal. (1990) J.Mol.Biol.215 (3): 403-410).In addition, percent homology between two amino acid sequences can also use Needleman and Wunsch algorithm to determine, wherein said algorithm is introduced in the GAP program of GCG software package, which use Blossum62 matrix or PAM250 matrix, and the gap weight of 16,14,12,10,8,6 or 4 and 1,2,3,4, the Length Weight (NeedlemanandWunsch, (1970) J.Mol.Biol.48:444-453) of 5 or 6.In addition, percent homology between two nucleotide sequences can also use the GAP program in GCG software package to determine, which use NWSgapdna.CMP matrix and 40,50,60,70 or 80 gap weight and 1,2,3,4, the Length Weight of 5 or 6.Arbitrary those of ordinary skill of this area can carry out initial homology and calculate, and correspondingly parameter is calculated in adjustment.Preferred parameter group (if with uncertain which parameter of should applying of operator to determine the parameter group whether molecule should use in the restriction of required homology) be have the gap penalty of 12, the gap extension penalties of 4 and 5 the Blossum62 rating matrix of frameshift gap point penalty.The additive method of sequence alignment is biological technical field known (for example, see Rosenberg (2005) BMCBioinformatics6:278); Altschuletal. (2005) FEBSJ.272 (20): 5101-5109).
" endogenous " polypeptide refers to the polypeptide by the genome encoding of host cell (such as parental generation microbial cell), and wherein recombinant cell is by described transform host cell or derivative to obtain.
" external source " polypeptide refers to not by the polypeptide of the genome encoding of parental generation microbial cell.Variant (that is, mutant) polypeptide is the example of allogenic polypeptide.
Term " allos " typically refers to be derived by different species and obtains or obtain by different organisms is derivative.As used herein, allos refer to and non-natural is present in nucleotide sequence in specific organism or peptide sequence.Heterogenous expression refers to does not express in the cell of this protein through testing to join usually by protein or polypeptide.Therefore, allos refer to such fact: the protein be transferred obtains by the cell dissimilar from acceptor or different species are derivative at first.Such as can be incorporated in bacterial host cell by the polynucleotide sequence endogenous for vegetable cell by recombination method, then this plant polynucleotides is heterologous polynucleotide in recombinant bacteria host cell.
As used herein, " fragment " of term polypeptide refers to that the shorter part of full-length polypeptide or protein, its size are that 4 amino acid residues to whole amino acid sequence deducts 1 amino acid residue.In some embodiment disclosed by the invention, fragment refers to the whole amino acid sequence of the domain (such as substrate-binding domain or catalyst structure domain) of polypeptide or protein.
As used herein, term " mutagenesis " refers to a kind of method, is changed in a stable manner by the organic hereditary information of the method.The mutagenesis of the nucleotide sequence of coded protein produces mutein.In addition, mutagenesis also refers to the change of non-coding nucleotide sequences, and this change can cause the protein active of modification.
As used herein, term " gene " refers to the nucleotide sequence of coding RNA product or protein; And affect the nucleotide sequence (such as this type of sequence includes but not limited to promoter or enhancer sequence) be operably connected or the nucleotide sequence that affect the sequence of RNA or protein expression, that be operably connected of encoding (such as this type of sequence includes but not limited to ribosome bind site or translation control sequence) of RNA or protein expression.
Expression control sequenc is known in the art, and comprise such as promoter, enhancer, polyadenosine acid signal, transcription terminator, internal ribosome entry site (IRES) etc., they provide the expression of polynucleotide sequence in host cell.Expression control sequenc specifically with transcribe relevant cell protein and interact (Maniatisetal. (1987) Science236:1237-1245).Described in exemplary expression control sequenc such as Goeddel, GeneExpressionTechnology:MethodsinEnzymology, Vol.185, AcademicPress, SanDiego, Calif. (1990).
In method disclosed by the invention, expression control sequenc is operably connected with polynucleotide sequence." be operably connected " and refer to that polynucleotide sequence is connected by this way with expression control sequenc, make suitable molecule (such as transcription activating protein white matter) and expression control sequenc in conjunction with time, allow gene expression.According to the direction of transcribing and translating, the promoter be operably connected is positioned at the upstream of selected polynucleotide sequence.The enhancer be operably connected can be positioned at the upstream of selected polynucleotide, inside or downstream.
As used herein, term " carrier " refers to the nucleic acid molecules that can transport connected another kind of nucleic acid (that is, polynucleotide sequence).The useful carrier of one type is episome (that is, can at the nucleic acid of extrachromosomal replication).Useful carrier for connected nucleic acid can be made independently to copy and/or express those.The carrier of the gene orientation expression be operably connected with it can be made also to be called in the present invention " expression vector ".In general, be generally " plasmid " form for the expression vector in recombinant DNA technology, it typically refers to the DNA circle of ring-type double-strand, and this DNA circle can not be combined with chromosome as carrier format.Term " plasmid " and " carrier " can exchange use in the present invention, because plasmid is the most frequently used carrier format.But also comprise this type of other forms of expression vector, these carriers play function of equal value and are up to the present known in the art.In some embodiments, recombinant vector comprises the promoter be operably connected with polynucleotide sequence further.In some embodiments, promoter be Growth adjustment, organelle-specificity, the promoter of tissue-specific, induction type, formation type or cell-specific.Recombinant vector generally includes at least one sequence, and it comprises the expression control sequenc that (a) operationally puts together with polynucleotide sequence; B selected marker that () and polynucleotide sequence are operationally puted together; C flag sequence that () and polynucleotide sequence are operationally puted together; D purification part that () and polynucleotide sequence are operationally puted together; E secretion sequence that () and polynucleotide sequence are operationally puted together; And the target sequence that (f) and polynucleotide sequence are operationally puted together.In certain embodiments, nucleotide sequence is stably incorporated in the genomic DNA of host cell, and the expression of nucleotide sequence is under the control of modulated promoter region.Expression vector of the present invention comprises the form being applicable to express polynucleotide sequence in host cell, polynucleotide sequence of the present invention.What it should be appreciated by those skilled in the art is the factor that the design of expression vector can depend on the selection of host cell such as to be transformed, the expression of required polypeptide and so on.Expression vector of the present invention be directed in host cell, thus produces by the polypeptide of polynucleotide sequence coding of the present invention, comprises fused polypeptide.In prokaryotes (such as Escherichia coli), the gene of expression coded polypeptide is the most common is use the carrier comprising formation type or inducible promoter to implement, and wherein said promoter instructs the expression of fusion or non-fused polypeptide.The amino acid of some joins in the polypeptide of wherein encoding by fusion vector, usually joins amino terminal or the carboxyl terminal of recombinant polypeptide.This fusion vector usually play in following 3 kinds of objects one or more: (1) increases the expression of recombinant polypeptide; (2) dissolubility of recombinant polypeptide is increased; And (3) contribute to the purifying of recombinant polypeptide by playing the effect of part in affinity purification.Usually, in the carrier of amalgamation and expression, introduce proteoclastic cleavage site in fusion part and the junction of recombinant polypeptide.This makes after fusion polypeptide purification, and recombinant polypeptide is separated with fusion part.In certain embodiments, polynucleotide sequence disclosed by the invention is operably connected with the promoter derived by Phage T5.In certain embodiments, host cell is yeast cells, and expression vector is Yeast expression carrier.Example for the carrier of expressing in saccharomyces cerevisiae (S.cerevisiae) comprises pYepSecl (Baldarietal. (1987) EMBOJ.6:229-234), pMFa (Kurjanetal. (1982) Cell30:933-943), pJRY88 (Schultzetal. (1987) Gene54:113-123), pYES2 (InvitrogenCorp., SanDiego, and picZ (InvitrogenCorp. CA), SanDiego, CA).In other embodiments, host cell is insect cell, and expression vector is rhabdovirus expression vector.The baculovirus vector being used in marking protein in the insect cell (such as Sf9 cell) of cultivation comprises such as pAc series (Smithetal. (1983) Mol.CellBiol.3:2156-2165) and pVL series (Lucklowetal. (1989) Virology170:31-39).In another embodiment, polynucleotide sequence of the present invention can use mammalian expression vector to express in mammalian cell.Well known in the art for protokaryon and other suitable expression systems eukaryotic, for example, see Sambrooketal., " MolecularCloning:ALaboratoryManual, " reprints, ColdSpringHarborLaboratory, (1989).
As used herein; " acyl group-CoA " refers to and form acyl thioester between the carbonyl carbon of alkyl chain and the sulfydryl of the 4'-phosphopan tetheine sulfinyl part of coacetylase (CoA); it has following formula R-C (O) S-CoA, and wherein R is any alkyl with at least 4 carbon atoms.
As used herein, " acyl-acp " refers to and form acyl thioester between the carbonyl carbon of alkyl chain and the sulfydryl of the phosphopantetheinyl moeity of acyl carrier protein (ACP).Phosphopantetheinyl moeity is connected with the conserved serine residue on ACP by the effect of complete-acyl carrier protein synthase (ACPS) (a kind of Phosphopantetheinyl transferase) upon translation.In some embodiments, acyl-acp is the intermediate in the synthesis of completely saturated acyl-acp.In other embodiments, acyl-acp is the intermediate in the synthesis of undersaturated acyl-acp.In some embodiments, carbochain has about 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 or 26 carbon.Each of these acyl-acps is all the substrate of the enzyme they being converted into derivative of fatty acid.
As used herein, term " fatty acid " biosynthesis pathway " refer to the biosynthesis pathway producing fatty acid and derivant (comprising ω-hydroxylated derivative of fatty acid) thereof.Except the enzyme that fatty acid biosynthetic pathway has the derivative of fatty acid (such as ω-hydroxylated derivative of fatty acid) of required feature except the production comprising the present invention and discuss or polypeptide, other the enzyme with enzymatic activity or polypeptide can also be comprised.
As used herein, term " clone " typically refers to and originates from single common ancestor and be the consistent cell of heredity or one group of cell substantially with these ancestors, the bacterium of the cloning bacteria bacterium colony such as formed by single bacterial cell.
As used herein, term " culture " typically refers to the fluid nutrient medium comprising living cells.In one embodiment, culture comprises the cell of regenerative ratio in predetermined culture medium under controlled conditions, the recombinant host cell culture such as grown in the fluid nutrient medium comprising selected carbon source and nitrogen." cultivation " of verb form or " cultivation " of occlusion instigates recombinant host cell colony to grow under suitable conditions in liquid or solid nutrient culture media.In particular embodiments, cultivation refers to that substrate is converted into end-product with fermentation mode biology.The nutrient culture media cultivated is known, and the single composition of this type of culture medium can derive from commercial source, such as Difco tMand BBL tMtrade mark.In the example of an indefiniteness, water-based nutrient medium is " enriched medium " of the complex source comprising nitrogen, salt and carbon, such as YP nutrient culture media, and it comprises 10g/L peptone and the 10g/L yeast extract of this type of nutrient culture media.According to United States Patent (USP) 5,000,000; 5,028,539; 5,424,202; 5,482,846; 5,602,030; Method described in WO2010127318, the host cell of culture can be transformed in addition, thus effectively assimilates carbon and use cellulosic material as carbon source.In addition, in some embodiments, host cell is transformed thus is expressed invertase, and such sucrose can be used as carbon source.
As used herein, term " under the condition of the polynucleotide sequence of effective expression genetic modification " refers to any condition of the ω-hydroxy fatty acid derivative allowed needed for host cell production.Suitable condition comprises such as fermentation condition.
Term " recombinant microorganism organism " refers to that host cell is through genetic modification, makes some enzymatic activity in host cell be changed relative to parental cell or native host cell, add and/or delete.The host cell of genetic modification is the example of restructuring microbial organisms.So, " modification or the protein activity levels that changes " (such as the enzyme) in recombinant host cell refers to the difference of one or more features in the activity determined relative to parental generation or native host cell (wherein lacking identical modification).Usually, active difference is (such as recombinant host cell culture the comparing relative to corresponding wild-type host cells) that measure between recombinant host cell (having modified activity) with corresponding wild-type host cells (not having modified activity).The activity of modifying can be the result of following situation, such as: the amount of the protein expressed by recombinant host cell changes (number of copies of the DNA sequence dna of such as coded protein raise or reduce, the quantity of the mRNA transcripton of coded protein raises or the amount of protein translation that reduces and/or is formed by mRNA protein raises or the result of reduction); The change (such as primary structure changes, and as the change of the coded sequence of protein, it causes the change of substrate specificity, the change of seen kinetic parameter) of protein structure; And the stability of protein changes (rising of such as protein degradation or reduction).In some embodiments, polypeptide is mutant or the variant of any polypeptide of the present invention.In certain embodiments, for the coded sequence of polypeptide of the present invention be the codon optimized for the expression of concrete host cell.Such as in order at expression in escherichia coli, one or more codons (such as described in Grosjeanetal. (1982) Gene18:199-209) can be optimized.
As used herein, term " adjustment sequence " typically refers to the sequence of base in DNA, and this sequence is operably connected with the DNA sequence dna of coded protein, and wherein said base sequence finally controls protein expression.The example of sequence is regulated to include but not limited to RNA promoter sequence, transcription factor binding sequence, transcription terminator, transcription regulatory factor (such as enhancer element), the nucleotide sequence affecting rna stability and translational regulation sequence (such as ribosome bind site (the Shine-Dalgarno sequence in such as prokaryotes or the Kozak sequence in eucaryote), initiation codon, terminator codon).
Term " expression of change " and " expression of modification " can exchange use, and refer at identical conditions, compared with polynucleotide, polypeptide, metabolin or the concentration of product (such as ω-hydroxy fatty acid derivative) in corresponding wild-type cell, polynucleotide, polypeptide, metabolin or product exist with different concentration in the host cell of transformation.
As used herein, term " is tired " and is referred to the quantitative of the omega-3-fatty acids derivant that the host cell cultures of per unit volume is produced.Tire and can refer to the combination of concrete omega-3-fatty acids derivant or the omega-3-fatty acids derivant produced by given recombinant host cell culture.
As used herein, " productive rate of the ω-hydroxy fatty acid derivative produced by host cell " refers to that the carbon source of input is converted into the efficiency of product (such as ω-hydroxy fatty acid, α, ω-diacid etc.) in host cell.Productive rate can refer to the combination of concrete omega-3-fatty acids derivant or the omega-3-fatty acids derivant produced by given recombinant host cell culture.ω-hydroxy fatty acid derivative includes but not limited to ω-hydroxy fatty acid, comprises ω-hydroxyl free fatty acid; ω-hydroxy fatty acid methyl ester; ω-oxo fatty acid; ω-oxo fatty acid methyl ester; Dual-function compound, such as α, ω-diacid; α, omega-diol; α, ω-diester; ω-carboxylic fatty acids methyl ester; Omega-amino-fatty acid; With omega-amino-fatty acid methyl ester.
As used herein, term " throughput rate " refers to the host cell cultures production ω-hydroxy fatty acid derivative of per unit volume in time per unit or the quantitative of derivant.Throughput rate can refer to the combination of concrete omega-3-fatty acids derivant or the derivative of fatty acid produced by given recombinant host cell culture.
As used herein, term " glucose utilization rate " refers to the amount of the glucose that culture uses in time per unit, and it is reported as grams per liter/hour (g/L/hr).
As used herein, term " carbon source " refers to the substrate or compound that are suitable for use as protokaryon or the carbon source needed for simple eukaryotic growth.Carbon source can be various ways, includes but not limited to polymkeric substance, carbohydrates, acid, alcohol, aldehyde, ketone, amino acid, peptide and gas (such as CO and CO 2).Exemplary carbon source includes but not limited to: monose, such as glucose, fructose, mannose, galactose, wood sugar and arabinose; Oligosaccharides, such as FOS and galactooligosaccharide; Polysaccharide, such as starch, cellulose, colloid and xylan; Disaccharides, such as sucrose, maltose, cellobiose and turanose; Cellulosic material and variant, such as hemicellulose, methylcellulose and sodium carboxymethyl cellulose; Succinate, lactate and acetate; Alcohol, such as ethanol, methyl alcohol and glycerine; Or their potpourri.In addition, carbon source can also be photosynthate, such as glucose.In certain embodiments, carbon source is living beings.In other embodiments, carbon source is glucose.In other embodiments, carbon source is sucrose.
When term " derives from the carbon source of reproducible feed " and is used alone or use with reference to feed source, its refer to comprise except grease chemical article (namely, the refining oil obtained by plant and animal, such as fatty acid, fatty acid ester, TAG, hydroxy fatty acid etc.) and petroleum chemicals is (namely, such as, derived from the chemicals of oil, alkane, alkene etc.) beyond, any biomaterial that can derive carbon (comprising reproducible feed and/or living beings and/or waste product).Therefore, as used herein, term " carbon source derived from reproducible feed " eliminates the carbon derived from grease chemical article and petroleum chemicals.In some embodiments, carbon source comprises sugar or carbohydrates (such as monose, disaccharides or polysaccharide).In some embodiments, carbon source is glucose and/or sucrose.In other embodiments, carbon source derived from reproducible feed, such as, derives from the carbohydrates of corn, sugarcane or lignocellulose biomass; Waste product, such as glycerine, waste gas, synthetic gas; The organic material improved, such as living beings or rock gas; Or carbon source is the carbon dioxide fixing by light compositing.In some embodiments, be suitable for the biological living beings transformed and be processed to carbon source.In other embodiments, living beings do not need to be processed into carbon source further, and directly can be used as carbon source.The exemplary source of this biolobic material is plant material or vegetables, such as switchgrass.Another kind of exemplary carbon source comprises metabolic waste, such as animal substance (such as cow dung just).Other exemplary carbon source comprises marine alga and other thalassophytes.Another kind of carbon source (comprising living beings) comprises the refuse obtained by industry, agricultural, forestry and family, includes but not limited to fermentation waste, fermentation of biomass, glycerin/glycerol, fresh stover, straw, timber, sewage, rubbish, independent solid waste, cellulose municipal waste and leftovers.
As used herein, the term " separation " of product (such as ω-hydroxy fatty acid derivative) is referred to and is separated by cell component, cell culture growth medium or precursor that is chemical or synthesis the product obtained.ω-the hydroxy fatty acid derivative produced by the method for the invention and ω-hydroxy fatty acid derivative composition can be relative immiscible in fermentation liquor and tenuigenin.Therefore, described ω-hydroxy fatty acid derivative and and ω-hydroxy fatty acid derivative composition can in cell or extracellular be collected in organic phase.In a preferred method, ω-hydroxy fatty acid derivative and and ω-hydroxy fatty acid derivative composition collect in extracellular.
As used herein, " purifying " of term verb, describe that " purifying " or nominal " purifying " of part of speech refers to and molecule shifted out from its environment by being such as separated or isolating (separation) or is separated." substantially purifying " molecule at least about 60% not containing (such as at least about 70% not containing, at least about 75% not containing, at least about 85% not containing, at least about 90% not containing, at least about 95% not containing, at least about 97% not containing, at least about 99% not containing) other compositions associated by them.As used herein, these terms also refer to remove pollutant from sample.The removal of such as pollutant can make the percent of derivative of fatty acid in sample increase.Such as when producing ω-hydroxy fatty acid derivative in recombinant host cell, purifying ω-hydroxy fatty acid derivative can be carried out by removing host cell proteins matter.After purifying, in sample, the percent of ω-hydroxy fatty acid derivative increases." purifying " of term verb, " purifying " or nominal " purifying " that describe part of speech are relative terms, and it does not need absolute purity.Therefore, such as when producing ω-hydroxy fatty acid derivative in recombinant host cell, the ω-hydroxy fatty acid derivative of purifying is basic and the basic ω-hydroxy fatty acid derivative of isolating of other cell components (such as nucleic acid, polypeptide, lipid, carbohydrates or other hydrocarbons).
The term used in instructions and claims " produce ω-hydroxy fatty acid derivative in vivo " and refer to live and/or genetic modification host cell in by the reproducible feed of such as carbohydrates and so on to produce ω-hydroxy fatty acid derivative, wherein said reproducible feed adds in fermentation liquor as carbon source, and such host cell can absorb and metabolism carbon source during the fermentation.These are different from the method producing ω-hydroxy fatty acid derivative in vitro, in the method, use enzyme or the cell lysate of purifying, and add in the enzyme of purifying for the direct substrate (such as fatty acid or derivative of fatty acid) of enzymatic conversion or add in cell lysate solution.In addition, method of the present invention is also different from the method wherein producing ω-hydroxy fatty acid derivative in biology transforms, in the method, use rest cell, and add in rest cell for source side formula beyond the direct substrate (such as fatty acid or derivative of fatty acid) of enzymatic conversion.
The transformation of approach and the activity of enzyme
Fatty acid synthesis is one of the most conservative system of bacterium living beings synthesis machine.Fatty acid synthase (FAS) multienzyme complex is present in all bacteriums and eukaryotic.Most FAS related gene is absolutely necessary for Growth of Cells and existence.Eucaryon and bacterium FAS drive the biochemical conversion of identical type in essence.In eukaryotic, FAS is called FASI, and its most catalyst structure domain is encoded by a polypeptied chain (inseparable).In the prokaryotes of such as bacterium and so on, FAS is called FASII, and its single enzyme and carrier protein are by the separately coded by said gene of (separation) protein of encoded discrete.So, FASII is the composite system with obviously change and different qualities.
Acyl carrier protein (ACP) controls the length of the fatty acid produced in native organism, saturation degree and branching together with the enzyme in FAS approach.Step in this approach synthesizes the enzyme institute catalysis of (FAB) and acetyl-CoA carboxylase (ACC) gene family by fatty acid biological.The such as enzyme that can be included in FAS approach comprises AccABCD, FabD, FabH, FabG, FabA, FabZ, FabI, FabK, FabL, FabM, FabB and FabF.According to required product, can weaken or one or more in these genes of process LAN.Like this, prokaryotes are through transforming thus adding the production being formed derivative of fatty acid by reproducible feed (such as glucose or other carbon sources).In the present invention, main target is the activity increasing key control enzyme (it regulates the production of derivative of fatty acid), thus bacterial isolates is changed into the microbiological plants produced for derivative of fatty acid (comprising fatty acid methyl ester (FAME), fatty-acid ethyl ester (FAEE) and fatty alcohol (FALC)) (for example, see U.S. Patent No. 8,283,143, the document is incorporated herein by reference).
The present invention openly identifies the polynucleotide of the polypeptide of codase function, thus has modified the enzymatic pathway for the production of required compound (such as ω-hydroxy fatty acid derivative).These polypeptide identified by enzyme accession number (EC numbering) in the present invention may be used for transforming fatty acid pathway, and this can produce bifunctional molecule, such as ω-hydroxy fatty acid derivative.Fig. 1-4 depicts the approach producing these compounds through transformation.Fig. 5 depicts the approach forming lactams and polymkeric substance (such as nylon).
In one embodiment, Fig. 1 to 4 depicts and uses the reproducible feed of such as glucose and so on to produce the approach of ω-hydroxy fatty acid derivative.Glucose changes into acyl-acp (step 1 see in figure to 4) by native organism.The polynucleotide that coding has the polypeptide of Fatty acid degradation enzymatic activity optionally can weaken (embodiment that vide infra) according to required product.The non-limiting example of this type of polypeptide is acyl-CoA synthase (FadD) and acyl-CoA dehydrogenase (FadE).What table 1 provided enzymatic activity (seeing below) in metabolic pathway enumerates thing in a large number, comprise the multiple Fatty acid degradation enzyme that optionally can weaken according to methods known in the art (for example, see U.S. Patent No. 8,283,143, see below).
Such as FadR is (see table 1, see below) be the important regulatory factor (Cronanetal. relevant with Fatty acid degradation and fatty acid biosynthetic pathway, Mol.Microbiol., 29 (4): 937-943 (1998)).Escherichia coli enzyme FadD (see table 1, seeing below) and fatty acid transport the component that protein FadL is fatty acid shooting system.FadL has mediated the transport of fatty acid to bacterial cell, and FadD has mediated the formation of acyl group-CoA ester.When not having other carbon sources to utilize; Exogenous Fatty Acid is taken in by bacterium and is changed into acyl group-CoA ester; it can be combined with transcription factor FadR; and suppress the expression of fad gene; described fad gene code is responsible for fatty acid and is transported (FadL), activates the protein of (FadD) and beta oxidation (FadA, FadB and FadE).When utilizing alternative carbon source, bacterium synthetic fatty acid is as acyl-acp, and it synthesizes for phosphatide, but can not as the substrate of beta oxidation.Therefore, acyl group-CoA and acyl-acp are all the independently sources of fatty acid, and it can obtain different end-products (Cavigliaetal., J.Biol.Chem., 279 (12): 1163-1169 (2004)).
Table 1: the activity of enzyme
The example of table 2A: ω-hydroxylase/omega oxidation enzyme (EC1.14.15.3)
Table 2B: for the example of the companion of ω-hydroxylase/omega oxidation enzyme (EC1.14.15.3) redoxomorphism
Title/name Organism Accession number #
Ferredoxin, ferredoxin reductase The bacterial classification OC4 of acinetobacter BAE78451,BAE78453
Ferredoxin, ferredoxin reductase Mycobacterium marinum M YP_001851444,YP_001851442
Ferredoxin, ferredoxin reductase The extra large bacillus VT8 of water oil YP_957887,YP_957889
alkG,alkT Pseudomonas putida GPo1 CAB54052,CAB54063
rubA,rubB Acinetobacter baylyi ADP1 CAA86925,CAA86926
Table 2C: the example of self-centered ω-1, ω-2, ω-3-hydroxylase/oxidase (EC1.14.14.1) fused protein
Table 2D: the example of the PFOR fused protein that self-centered I class P450 merges
Table 3A: the example of alcohol dehydrogenase (EC1.1.1.1/2) or alcohol oxidase (EC1.1.3.13, EC1.1.3.20)
Title/name Organism Accession number #
alkJ Pseudomonas putida GPo1 CAB54054
alkJ Bo Ku island alkane eating bacteria (Alcanivorax borkumensis) AP1 CAC38030
cddC Rhodococcus ruber (Rhodococcus ruber) SC1 AAL14237
Table 3B: the example of aldehyde dehydrogenase (EC1.2.1.3/4/5/) or aldehyde oxidase (EC1.2.3.1)
Title/name Organism Accession number #
alkH Pseudomonas putida GPo1 CAB51050
alkH Bo Ku island alkane eating bacteria AP1 CAC38029
cddD Rhodococcus ruber SC1 AAL14238
Table 4: the example of aminopherase/transaminase (EC2.6.1) and amine dehydrogenase (EC1.4.9, EC1.4.98, EC1.4.99)
Table 5: the example of esterase (EC3.1.1.1) and lipase (EC3.1.1.3)
Title/name Organism Accession number #
lipA Pseudomonas fluorescens B52 AAF80996
phaZ Pseudomonas fluorescens GK13 AAA64538
Table 6: the example of hydrolytic enzyme (EC3.5.2.12)
Title/name Organism Accession number #
Omega-lauric lactam hydrolytic enzyme The t bacteria 31 of Acidovorax BAH09870
Omega-lauric lactam hydrolytic enzyme The bacterial classification U124 of greedy copper Pseudomonas BAH09871
Table 7: the example of acyl-CoA synthase/acyl group-CoA ligase (EC6.2.1.3)/transferase (EC2.8.3.6)
Table 8: the example of amide synthase
Title/name Organism Accession number #
Palmityl putrescine synthase (PPS) Not cultured bacterium AAV33349
N-(4-amino-2-hydroxybutyl) meat all can amide synthase (AhtS) Not cultured bacterium ACX33975
Table 9: the example of ester synthase (EC2.3.1.75 or EC2.3.1.20)
Title/name Organism Accession number #
AtfA The bacterial classification ADP1 of acinetobacter Q8GGG1
AtfA1 Bo Ku island alkane eating bacteria SK2 YP_694462
AtfA2 Bo Ku island alkane eating bacteria SK2 YP_693524
WS/DGAT Marinobacter alginolyticus WP_007153340
WS/DGAT The bacterial classification MED105 of phenol degrading Pseudomonas WP_008251579
ES9 Except hydrocarbon sea bacillus ABO21021
Fig. 1 and 2 shows wherein acyl-acp can change into α by 2 similar approach; the approach of ω-diacid; 2 wherein said approach use C12 free fatty acid (FFA) as precursor intermediate (see Fig. 1) respectively, or C12 fatty acid methyl ester (FAME) is as intermediate (see Fig. 2 A and 2B).
In one embodiment, Fig. 1 shows the production of number of chemical compound, comprises ω-hydroxy fatty acid, ω-oxo fatty acid, α, ω-diacid and omega-amino-fatty acid.In the step 2 of Fig. 1, use thioesterase that acyl-acp is changed into FFA.In certain embodiments, the gene of coding thioesterase is tesA, ' tesA, tesB, fatB1, fatB2, fatB3, fatA1 or fatA.(see also table 1, it illustrates the polypeptide of the enzymatic activity with thioesterase, wherein said thioesterase may be used for this step of catalysis, as mentioned above).In step 3, be also called that the ω-hydroxylase of omega oxidation enzyme is for generating ω-hydroxy fatty acid.As shown in Figure 1, the omega position of fatty acid is hydroxyl.
The CYP153A-reductase hybrid fusion polypeptide of expressing in recombinant host cell
The example of suitable ω-hydroxylase/omega oxidation enzyme (EC1.14.15.3) and their redox partners is listed in table 2A and 2B (as mentioned above).These are some non-heme ferrous oxidase (such as deriving from the alkB of pseudomonas putida GPo1), or some protoheme type P450 oxidase (such as deriving from the cyp153A of the water extra large bacillus of oil (Marinobacteraquaeolei)), also referred to as Cytochrome P450.Cytochrome P450 is the enzyme of extensively distribution, and it has the complicacy of height, and shows the activity of wide spectrum.They are by the multiple substrate of conversion and the protein coded by gene superfamilies of catalysis number of chemical reaction.Cyp153A is the superfamily of soluble bacterial Cytochrome P450, and it is to have the selectivity dihydroxy hydrocarbon chain (vanBeilenetal. (2006) Appl.Environ.Microbiol.72:59-65) of height to ω-position.The member of cyp153A family demonstrates optionally hydroxylation alkane in vitro, ω-the position of fatty acid or fatty alcohol, described member is such as deriving from the cyp153A6 (Funhoffetal. (2006) J.Bacteriol.188:5220-5227) of Mycobacterium species HXN-1500, the cyp153A16 deriving from Mycobacterium marinum and the cyp153A deriving from polar region zygosaccharomyces bacterial classification JS666 (Schepsetal. (2011) Org.Biomol.Chem.9:6727-6733), and derive from the cyp153A (Honda-Malcaetal. (2012) Chem.Commun.48:5115-5117) of the extra large bacillus of water oil.
As all Cytochrome P450s, Cyp153A ω-hydroxylase needs electronics for their catalytic activity, and these electronics are provided by specific redox protein matter, such as ferredoxin and ferredoxin reductase.These are interactional discrete protein with cyp153A.Before, by will cyp153A (Kubotaetal. (2005) Biosci.Biotechnol.Biochem.69:2421-2430 of Bo Ku island alkane eating bacteria SK2 be derived from; Fujitaetal. (2009) Biosci.Biotechnol.Biochem.73:1825-1830) (it comprises flavin mononucleotide (FMN) (FMN) and NADPH binding site with the reductase domain deriving from P450RhF, and [2FeS] ferredoxin center (Hunteretal. (2005) FEBSLett.579:2215-2220)) merge and create self-centered hybridization (being fitted together to) cyp153A oxidase (that is, its activity does not need the oxidase of discrete ferredoxin and ferredoxin reductase protein).P450RhF belongs to the PFOR (DeMotandParret (2003) TrendsMicrobiol.10:502) that I class P450 merges.This hybridization cyp153A-RedRhF fused protein in vitro biological transform in be presented at ω-position hydroxylation octane, and other compound of hydroxylation, such as cyclohexane or butyl benzene.The example of natural P450-reductase fused protein is shown in table 2C and 2D (as mentioned above).
Consider that the ω-position of the oxidase of cyp153A family to hydrocarbon chain has the selectivity of height, it is shown as and can be used for by reproducible carbon source to produce α, the good example of the suitable candidate of ω-difunctional derivative of fatty acid.This allows the method for research and development commericially feasible to produce these valuable compounds therefroms.But, as other Cytochrome P450, up to the present the protein of cyp153A family be mainly used in in vitro test, these tests employ enzyme or the cell lysate crude product of purifying, or the biology being in rest cell transforms (Kubotaetal. in (source side formula adds derivative of fatty acid or hydrocarbon wherein), Fujitaetal., Honda-Malcaetal., as mentioned above).But, use the extracorporeal procedures of hybrid fusion or the conversion of rest cell biology can not implement also cost on a large scale and effectively produce ω-hydroxy fatty acid derivative.The knowledge accepted extensively in this area is that many Cytochrome P450s and alkB type ω-hydroxylase are not easy functionally to express in recombinant microorganism organism, this is because these enzymes normally non-activity, and their chemical action is difficult to illustrate.In fact, current trial uses the interior work of the unique body being different from the renewable carbon source of derivative of fatty acid to employ alkB ω-hydroxylase, and in the fermentation of high-cell density, only achieve the ω-hydroxy fatty acid derivative (WO2013/024114A2) of low liter.
The applicant creates CYP153A-reductase hybrid fusion protein matter and variant thereof, and they can produce ω-hydroxy fatty acid derivative by reproducible carbon source in vivo effectively.More specifically, the gene fusion (see embodiment 6, vide infra) containing c-end FMN-and Fe/S reductase domain of gene and the coding P450RhF from the bacterial classification NCIMB9784 of Rhod from the extra large bacillus of water oil of the hybrid fusion protein matter (wherein the alanine (A) at glycocoll (G) the position of substitution 307 place) of coding CYP153A (G307A) P450 catalyst structure domain.The polypeptide of gained is CYP153A-RedRhF hybrid fusion polypeptide (SEQIDNO:6), has corresponding nucleotide sequence (SEQIDNO:5).When this CYP153A-reductase hybrid fusion protein matter (uses simple carbon source at Bacillus coli cells, such as glucose) in express time, derivative of fatty acid can change into ω-hydroxy fatty acid derivative (see embodiment, vide infra) effectively.Suitable ω-hydroxylase (EC1.14.15.3) is listed in table 2A and 2B (as mentioned above) with other examples of their redox partners (may be used for generating similar CYP153A-reductase hybrid fusion polypeptide).
Present disclosure provides the microbial organisms effectively and optionally can producing ω-hydroxy fatty acid derivative (comprising α, ω-difunctional derivative of fatty acid) in vivo.In one embodiment, CYP153A-RedRhF hybrid fusion protein matter is expressed in microbial organisms through transformation, makes described microbial organisms effectively the converting compounds of such as cinnamic acid or dodecylic acid methyl ester and so on can be become sabinic acid or sabinic acid methyl ester by carbon source (such as glucose) in vivo.Any reproducible feed can replace glucose and be used as carbon source.Therefore, (namely first time display works as the hybrid fusion protein matter of the transformation with P450 enzymatic activity, illustrated by CYP153A-RedRhF hybrid fusion protein matter) with thioesterase coexpression when feeding carbon source by reproducible feed in the host cell (such as Escherichia coli), described hybrid fusion protein matter can effectively convert fatty acids be become in vivo specific needed for ω-hydroxylated compound (such as ω-hydroxy fatty acid; ω-hydroxy fatty acid methyl ester; α, ω-hydroxy dibasic acid; α, ω-hydroxyl diester; And α, omega-diol) (see embodiment 6 and Figure 25 and 26).Open according to the present invention, can by P450 gene (gene of cyp153A protein of such as encoding) be connected with reductase gene (the c-end reductase domain of the PFOR protein that the I class P450 that such as encodes merges or other domain) the hybrid fusion protein matter transforming other.Table 2A and 2D (as mentioned above) sets forth the example of the PFOR protein that cyp153A and I class P450 merges.Instruct according to these, similar fused protein can be created by the ω-hydroxylase/omega oxidation enzyme of other types.
As shown in Figure 2 A, α is formed by ω-hydroxy fatty acid methyl ester, the approach of ω-diacid or omega-amino-fatty acid, or as shown in Figure 2 B, pass through α, omega-3-fatty acids dimethyl esters forms α, the approach of ω-diacid is favourable, this is because methyl ester is uncharged, this provide the advantage of large-scale production and recovery.In addition, allow by the approach of ω-hydroxy fatty acid methyl ester the lactamize being carried out direct enzyme by the catalysis of some hydrolytic enzyme.In the step 3 of Fig. 1 or Fig. 2 A and 2B, ω-hydroxy fatty acid can be changed into ω-oxo fatty acid and (such as show the polypeptide that 3A display has alcohol dehydrogenase or oxidasic enzymatic activity by alcohol dehydrogenase or oxidase further, wherein said alcohol dehydrogenase or oxidase may be used for this step of catalysis, as mentioned above).Alcohol oxidase (the flavoprotein that the suitable enzyme that such as sabinic acid or sabinic acid methyl ester can be oxidized to 12-oxo dodecylic acid or 12-oxo dodecylic acid methyl ester is, such as derive from the alkJ of pseudomonas putida) (EC1.1.3.13, EC1.1.3.20) alcohol dehydrogenase (such as deriving from the cddC of Rhodococcus ruber) (EC1.1.1.1) (see table 3A, as mentioned above) that relies on of (see SEQIDNO:67) or NAD (P).Thus, described approach thus can according to 2 kinds of different alternative route, namely, by aldehyde dehydrogenase or oxidase, ω-oxo convert fatty acids is become α, ω-diacid (such as shows the polypeptide that 3B display has aldehyde dehydrogenase or oxidasic enzymatic activity, wherein said aldehyde dehydrogenase or oxidase may be used for this step of catalysis, as mentioned above), or pass through aminopherase, ω-oxo convert fatty acids is become omega-amino-fatty acid by transaminase or amino acid dehydrogenase, and (such as table 4 display has aminopherase, the polypeptide of the enzymatic activity of transaminase or amino acid dehydrogenase, wherein said aminopherase, transaminase or amino acid dehydrogenase may be used for this step of catalysis, as mentioned above).Suitable transaminase such as 12-oxo dodecylic acid or 12-oxo dodecylic acid methyl ester being changed into 12 amino dodecanoic acid or 12-amino methyl ester is shown in Table 4 (as mentioned above).Amido is transferred to ω-oxyacid or ester by suitable donor by these enzymes, thus generates corresponding last amine (terminalamine) eventually.The coexpression of several amino acids dehydrogenasa (such as alanine dehydrogenase, glutamate dehydrogenase, see table 4, as mentioned above) can be passed through and improve the utilizability of amine donor (such as alanine or glutamate) in vivo.In addition, the homologue of glutamate dehydrogenase (NADH can be utilized to replace NADPH) or the coexpression of other variants can be provided for the extra improvement of production.In addition, the example being applicable to the enzyme (such as methyl amine dehydrogenasa or lysine 6-dehydrogenasa) 12-oxo dodecylic acid or 12-oxo dodecylic acid methyl ester being changed into other kinds of 12 amino dodecanoic acid or 12-amino methyl ester is also listed in table 4.
In another embodiment, Fig. 2 A and 2B show by methyl ester as intermediate production number of chemical compound, comprise α, ω-diacid and omega-amino-fatty acid.The step 6 of Fig. 2 B shows α, the production (using acyl group-CoA ligase/transferase) of ω-diester.In the step 2 of Fig. 2 A and 2B, ester synthase is used acyl-acp to be changed into fatty acid methyl ester (FAME).In certain embodiments, the gene of coding ester synthase is the gene that codase is categorized as the enzyme of EC2.3.1.75 or EC2.3.1.20, coding wax/dgat (derives from Jojoba, the bacterial classification ADP1 of acinetobacter, Bo Ku island alkane eating bacteria, Pseudomonas aeruginosa, Fundibacterjadensis, difunctional ester synthase/acyl group-the CoA of arabidopsis or Alkaligeneseutrophus: diacylglycerol acyltransferase) gene, or coding AtfA1, AtfA2, ES9, the gene of ES8 or their variant (in addition, see table 9, it illustrates the polypeptide of the enzymatic activity with ester synthase, wherein said ester synthase may be used for this step of catalysis, as mentioned above).In step 3, (such as show 2A and show the polypeptide with enzymatic activity, this enzymatic activity may be used for this step of catalysis, as mentioned above) to use ω-hydroxylase (or omega oxidation enzyme) to generate ω-hydroxy fatty acid methyl ester.As visible in Fig. 2 A, the omega position of fatty acid methyl ester is hydroxyl.In the step 3 of Fig. 2 A, alcohol dehydrogenase or oxidase may be used for ω-hydroxy fatty acid methyl ester being changed into ω-oxo fatty acid methyl ester and (such as show 3A and show the polypeptide with alcohol dehydrogenase or oxidasic enzymatic activity, wherein said alcohol dehydrogenase or oxidase may be used for this step of catalysis, as mentioned above).Similarly, described approach thus can according to 2 kinds of different alternative route, namely, a kind of approach by aldehyde dehydrogenase or oxidase (see table 3B, as mentioned above) and final esterase or lipase (see table 5, as mentioned above) or by chemical conversion ω-oxo fatty acid methyl ester is changed into α, ω-diacid; And another kind of approach passes through aminopherase or transaminase or amino acid dehydrogenase (see table 4, as mentioned above) and final esterase or lipase (see table 5, as mentioned above) ω-oxo fatty acid methyl ester is changed into omega-amino-fatty acid.Final step (that is, wherein esterase or lipase-catalyzed by α, omega-3-fatty acids methyl ester or omega-amino-fatty acid methyl ester change into shown final product) can be the result of enzymatic conversion step and chemical conversion steps.Such as can the suitable enzyme of this step of catalysis list in table 5.Selected lipase (EC3.1.1.3) or esterase (EC3.1.1.1) are positioned at pericentral siphon, or secretion is in supernatant.Suitable secretion signal or anchor series can be transformed according to methods known in the art.In another embodiment, Fig. 2 B shows by α, and omega-3-fatty acids dimethyl esters produces α, ω-diacid.This approach is similar to the left side in Fig. 2 A, but it uses other acyl-CoA synthase/acyl group-CoA ligase or transferase (for suitable material standed for, see table 7, described above).In addition, the ester synthase/acyltransferase of catalysis first step can also catalysis step second from the bottom.
In another embodiment, Fig. 3 and 4 depicts and uses the carbon source (such as glucose) obtained by reproducible feed to produce α by the fatty alcohol produced in cell (FALC), the approach of omega-diol.In one embodiment, Fig. 3 shows wherein by using thioesterase or carboxylate reductase activity to prepare α, and the approach of omega-diol (for can the polypeptide of these steps of catalysis, see table 1, as mentioned above).In the present invention, in step 2, thioesterase can change into free fatty acid (FFA) by catalyzing acyl-ACP.In step 3, the FFA of gained can be changed into fatty aldehyde by carboxylate reductase.In step 4, fatty aldehyde (see table 1, can be changed into fatty alcohol (FALC) by alcohol dehydrogenase as mentioned above).Finally, in steps of 5, FALC (see table 2A, can be changed into α, omega-diol by ω-hydroxylase or omega oxidation enzyme as mentioned above).Alternatively, acyl-acp can directly change into fatty aldehyde by acyl-acp reductase, as shown in Figure 4.(table 1 shows the polypeptide of the enzymatic activity with thioesterase, carboxylate reductase, acyl-acp reductase or alcohol dehydrogenase, thus these steps of catalysis, as mentioned above).
In another embodiment, show the approach producing lactams, described lactams chemically can change into polymkeric substance.This is shown in Figure 5 with the example by omega-amino-fatty acid (such as 12 amino dodecanoic acid); wherein said omega-amino-fatty acid passes through acyl group-CoA-transferase (see table 7; as mentioned above) and amide synthase (see table 8, as mentioned above) and change into lactams.In this concrete example, the lactams of gained is 12-aminododecane lactams or lauric lactam.The example of the polymkeric substance derived by it is nylon 12, as shown in Figure 5.For direct for 12 amino dodecanoic acid methyl ester lactams being changed into the suitable enzyme of corresponding lactams for some hydrolytic enzyme is (see table 6, as mentioned above), it can combinationally use with other enzymes in identical biocatalyst, thus instructs the production of lauric lactam; Or during the biology separated that may be used for 12 amino dodecanoic acid methyl ester transforms.Direct for 12 amino dodecanoic acid methyl ester lactams (see table 5, can also be changed into corresponding lactams by lipase or esterase as mentioned above).
The present invention openly identifies the polynucleotide that coding has the polypeptide of enzymatic activity, and wherein said polypeptide may be used in recombinant host cell with in production method.The polypeptide with enzymatic activity contributes to producing the composition comprising described compound.Universally recognized is without the need to the absolute sequence identity with these type of polynucleotide.Specific polynucleotide sequence (such as coding has the polynucleotide of the polypeptide of enzyme function) such as can be made to change, and screen coded polypeptide for activity.This type of change comprises conservative sudden change and silent mutation (such as codon optimized) usually.Methods known in the art can be used, polynucleotide that are that carry out screening-gene transformation for required function or that modify and coded variant polypeptide, wherein said function includes but not limited to the catalytic activity of the increase increased, is increased to the suppression (such as feeding back the suppression of reduction) of stability or reduction.
In addition, the present invention is open to number according to enzyme classification (EC), identify and (relate to the production of ω as herein described-hydroxylated derivative of fatty acid with the approach through transforming, multiple steps as mentioned above) (namely, reaction) relevant enzymatic activity, and provide and numbered by this type of EC and the Exemplary polynucleotide of the Exemplary polypeptide (such as enzyme) of classifying and encoding such polypeptides.This type of Exemplary polypeptide and polynucleotide are (in the present invention, it is identified by accession number and/or sequence identification number (SEQIDNO)) for transforming the fatty acid pathway in parent host cell, this approach can produce ω-hydroxy fatty acid derivative, comprise other bifunctional molecules, such as α, ω-diacid, thus the host cell obtaining genetic modification of the present invention.Polypeptide of the present invention and polynucleotide are exemplary and indefiniteness.Those skilled in the art can pass through multiple database (Entrez database that such as theNationalCenterforBiotechnologyInformation (NCBI) provides, the ExPasy database that theSwissInstituteofBioinformatics provides, the BRENDA database that theTechnicalUniversityofBraunschweig provides, and the KEGG database that theBioinformaticsCenterofKyotoUniversityandUniversityofT okyo provides, all these all can derive from internet) obtain the sequence of the homologue of Exemplary polypeptide of the present invention.
In one embodiment, recombinant microorganism organism (as mentioned above) of the present invention can produce the ω-hydroxy fatty acid derivative with even carbon chain.In another embodiment, described recombinant microorganism organism can be transformed, thus produces the ω-hydroxy fatty acid derivative with odd carbon chain.Such as ω-hydroxylase approach can be expressed in the recombinant cell (such as Escherichia coli) of derivative of fatty acid excessively producing odd number chain (the odd number chain fatty acid derivant excessively produced by some bacterial isolates have in U.S. Patent Application Publication US2012/0070868 described by, the document is incorporated herein by reference).This allows the ω-hydroxylated derivative of fatty acid producing odd number chain.In one embodiment, the recombinant bacterial strain of excessive production odd number chain fatty acid is when combining with expression ω-hydroxylase (such as CYP153A-RedRhF hybrid fusion protein matter) and decarboxylation of fatty acids enzyme (such as oleT) (for example, see Rudeetal. (2011) Appl.Environ.Microbiol.77:1718), produce the fatty alcohol with the even number chain of terminal double bond, such as 9-decen-1-ol, 11-dodecene-1-alcohol, 13-tetradecene-1-alcohol and 15-hexadecene-1-ol.
In another embodiment, the hydroxylated derivative of fatty acid of proximal end is provided.These compounds are in omega-1 position, and/or omega-2 position, and/or omega-3 position, and/or omega-4 position etc. (such as ω-1, ω-2 and/or ω-3 etc.) have the compound of at least one OH group.Embodiment 8 and 9 shows and derives from the Cytochrome P450 oxidase of cyp102A family by use and in the host cell of genetic modification, produce these compounds (Whitehouseetal. (2012) Chem.Soc.Rev.41:1218).The Cytochrome P450 oxidase of cyp102A family is applicable to produce the hydroxylated derivative of fatty acid of proximal end.But, these enzymes are not suitable for effectively produces ω-hydroxylated derivative of fatty acid in recombinant host, unless their substrate specificity is changed, make the derivative of fatty acid of their main hydroxylation ω-positions, up to the present, this is only part successful (Lentzetal. (2006) ChemBioChem.7:345).
The CYP153A-reductase hybrid fusion polypeptide variants of expressing in recombinant host cell
The present invention openly identifies CYP153A-reductase hybrid fusion polypeptide variants, when it expresses in recombinant host cell, obtains high tiring, the ω-hydroxylated fatty acid derived compositions of productive rate and/or throughput rate.In non-limiting example disclosed by the invention, CYP153A (G307A)-RedRhF hybrid fusion polypeptide is (see embodiment 6, vide infra) be used as effectively transforming the template of CYP153A-reductase hybrid fusion polypeptide variants (see embodiment 14-10, vide infra), thus the ω-OH fatty acid of production recruitment and ω-OH derivative of fatty acid.Such as when CYP153A-reductase hybrid fusion polypeptide variants is expressed in host cell, the converting compounds of such as dodecylic acid and so on can be become sabinic acid by simple carbon source (such as glucose) by effectively.Any simple carbon source (such as obtaining by reproducible feed is derivative) is all suitable.Show, when transformation CYP153A-reductase hybrid fusion polypeptide variants (namely, illustrated by the CYP153A-RedRhF hybrid fusion polypeptide variants of transformation) with thioesterase in the host cell (such as Escherichia coli) during coexpression, convert fatty acids can be become specific required compound (comprising ω-OH fatty acid) (see embodiment, vide infra) by utilizing the carbon source (such as glucose) that be obtained by reproducible feed by effectively.Open according to the present invention, can by the hybrid fusion polypeptide variants (see showing 2A to 2D, described above) that fetch and transform other that the gene (gene of CYP153A protein of such as encoding) of sudden change is connected with the gene of the sudden change of coding c-end reductase domain.Present invention encompasses multiple change, the sudden change (P5450 and reductase domain) of such as 2 genes or the sudden change (P450 or reductase domain) of 1 gene.Instruct according to these, similar fused protein variant can be created by the ω-hydroxylase of other types, and make them express in recombinant host cell, thus produce ω-OH derivative of fatty acid.
Therefore, the present invention openly relates to the recombinant host cell of expressing CYP153A-reductase hybrid fusion polypeptide variants, when use carbon source (such as glucose, sucrose or any other the carbon source derived by reproducible feed) cultivate described in cell time, its produce high to tire, the ω-hydroxylated fatty acid derived compositions of productive rate and/or throughput rate.CYP153A-reductase hybrid fusion polypeptide variants CYP153A domain or go back prodomain or both in there is one or more sudden change.In one embodiment, present disclosure provides CYP153A-reductase hybrid fusion polypeptide variants, itself and SEQIDNO:6 have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the sequence identity of 98% or 99%, and at amino acid position place, there is one or more sudden change, wherein said amino acid position comprises position 27, 82, 141, 178, 231, 309, 407, 415, 516, 666 and/or 796, wherein said CYP153A-reductase hybrid fusion polypeptide variants in vivo catalyze fatty acid changes into ω-OH fatty acid.More specifically, CYP153A-reductase hybrid fusion polypeptide variants has the one or more of following sudden change, comprising: R27L, and wherein arginine (R) replaces lysine (L); Position R82D, wherein arginine (R) replaces aspartic acid (D); Position V141I, wherein valine replaces isoleucine (I); Position V141Q, wherein valine (V) replaces glutamine (Q); Position V141G, wherein valine (V) substituted glycinic acid (G); Position V141M, wherein valine (V) replaces methionine (M); Position V141L, wherein valine (V) replaces leucine (L); Position V141T, wherein valine (V) replaces threonine (T); Position R178N, wherein arginine (R) replaces N (N); Position A231T, wherein alanine (A) replaces threonine (T); Position N309R, wherein N (N) replaces arginine (R); Position N407A, wherein N (N) substituted lactamine (A); Position V415R, wherein valine (V) replaces arginine (R); Position T516V, wherein threonine (T) replaces valine (V); Position P666A, wherein proline (P) substituted lactamine (A); Position P666D, wherein proline (P) replaces aspartic acid (D); And position A796V, wherein alanine (A) replaces valine (V).The example of CYP153A-reductase hybrid fusion polypeptide variants comprises SEQIDNO:8, SEQIDNO:10, SEQIDNO:12, SEQIDNO:14, SEQIDNO:16, SEQIDNO:18, SEQIDNO:20, SEQIDNO:22, SEQIDNO:24, SEQIDNO:26, SEQIDNO:28 or SEQIDNO:30, SEQIDNO:32, SEQIDNO:34, SEQIDNO:36, SEQIDNO:38, SEQIDNO:40, SEQIDNO:42, SEQIDNO:44 and SEQIDNO:46.In one embodiment, CYP153A-reductase hybrid fusion polypeptide variants is hybridization cyp153A-RedRhF-type fused protein variant.In another embodiment, the CYP153A-reductase hybrid fusion polypeptide variants in recombinant host cell obtains the ω-OH fatty acid derived compositions of the more high-titer of tiring than the ω-OH aliphatic acid composition produced by expressing CYP153A-reductase hybrid fusion polypeptide in corresponding host cell.In another embodiment, the CYP153A-reductase hybrid fusion polypeptide variants in recombinant host cell obtains the ω-OH fatty acid derived compositions of more high-titer, includes but not limited to ω-OHC 12, ω-OHC 14, ω-OHC 16, ω-OHC 18, ω-OHC 12:1, ω-OHC 14:1, ω-OHC 16:1with ω-OHC 18:1fatty acid derived compositions.
In another embodiment, CYP153A-reductase hybrid fusion polypeptide variants has sudden change at amino acid position 141 place, comprises V141I and/or V141T.In the present invention, the expression of CYP153A-reductase hybrid fusion polypeptide variants in recombinant host cell with sudden change V141I or V141T obtains respectively than the ω-OHC produced by expressing CYP153A-reductase hybrid fusion polypeptide 12or C 16ω-the OHC of the more high-titer of tiring of aliphatic acid composition 12or C 16aliphatic acid composition.In one embodiment, CYP153A-reductase hybrid fusion polypeptide variants has sudden change V141I and A231T (SEQIDNO:32), and when it expresses in the host cell of enzyme function with thioesterase, the ω-OHC of production recruitment 12fatty acid.In another embodiment, CYP153A-reductase hybrid fusion polypeptide variants has sudden change R27L, R82D, V141M, R178N and N407A (SEQIDNO:34), and when it expresses in the host cell of enzyme function with thioesterase, the ω-OHC of production recruitment 12fatty acid.In another embodiment, CYP153A-reductase hybrid fusion polypeptide variants has sudden change P666A (SEQIDNO:36), and when it expresses in the host cell of enzyme function with thioesterase, the ω-OHC of production recruitment 12fatty acid.In another embodiment, CYP153A-reductase hybrid fusion polypeptide variants has sudden change A796V (SEQIDNO:38), and when it expresses in the host cell of enzyme function with thioesterase, the ω-OHC of production recruitment 12fatty acid.In another embodiment, CYP153A-reductase hybrid fusion polypeptide variants has sudden change A796V, P666D and T516V (SEQIDNO:40), and when it expresses in the host cell of enzyme function with thioesterase, the ω-OHC of production recruitment 12fatty acid.In another embodiment, CYP153A-reductase hybrid fusion polypeptide variants has sudden change V141I, A231T and A796V (SEQIDNO:42), and when it expresses in the host cell of enzyme function with thioesterase, the ω-OHC of production recruitment 12and C 16fatty acid.In another embodiment, CYP153A-reductase hybrid fusion polypeptide variants has sudden change R27L, R82D, V141M, R178N, N407A and A796V (SEQIDNO:44), and when it expresses in the host cell of enzyme function with thioesterase, the ω-OHC of production recruitment 12fatty acid.In another embodiment, CYP153A-reductase hybrid fusion polypeptide variants has sudden change V141T, A231T and A796V (SEQIDNO:46), and when it expresses in the host cell of enzyme function with thioesterase, the ω-OHC of production recruitment 16fatty acid.
The present invention openly identifies the relevant polynucleotide of CYP153A-reductase hybrid fusion and polypeptide variants.CYP153A-reductase hybrid fusion polypeptide variants comprises SEQIDNO:8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44 and 46.CYP153A-reductase hybrid fusion Nucleic acid variant (DNA sequence dna) comprises SEQIDNO:7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45 and 47.But, be recognized that without the need to the absolute sequence identity with CYP153A-reductase hybrid fusion polynucleotide variant.Specific polynucleotide sequence such as can be made to change, and screen the polypeptide of coding for activity.This type of change generally includes conservative variants and silent mutation (such as by codon optimized).Methods known in the art can be used, for required function screen transformation or modify (namely, sudden change) polynucleotide and coded variant polypeptide, wherein said function is such as the function than wild type or template peptide improvement, includes but not limited to the catalytic activity of increase, the stability of increase or the suppression (feedback inhibition such as reduced) of reduction.The present invention is open to number according to enzyme classification (EC), identify with multiple steps of fatty acid biosynthetic pathway as herein described (namely, reaction) relevant enzymatic activity, and provide and numbered by this type of EC and the Exemplary polynucleotide of the Exemplary polypeptide (such as it plays the effect of specific enzyme and shows the activity of specific enzyme) of classifying and encoding such polypeptides.(in the present invention, it is by sequence identification number (SEQIDNO for this type of Exemplary polypeptide and polynucleotide; Identify as mentioned above)) for the fatty acid pathway in engineered host cell, such as, approach shown in Fig. 1.But it should be understood that polypeptide of the present invention and polynucleotide are exemplary, is therefore indefiniteness.Those skilled in the art can use multiple database (Entrez database that such as theNationalCenterforBiotechnologyInformation (NCBI) provides, the ExPasy database that theSwissInstituteofBioinformatics provides, the BRENDA database that theTechnicalUniversityofBraunschweig provides, and the KEGG database that theBioinformaticsCenterofKyotoUniversityandUniversityofT okyo provides, all these all can derive from internet) obtain the sequence of the homologue of Exemplary polypeptide of the present invention.
In one embodiment, for implementing CYP153A-reductase hybrid fusion polypeptide variants disclosed by the invention and SEQIDNO:8, SEQIDNO:10, SEQIDNO:12, SEQIDNO:14, SEQIDNO:16, SEQIDNO:18, SEQIDNO:20, SEQIDNO:22, SEQIDNO:24, SEQIDNO:26, SEQIDNO:28 or SEQIDNO:30, SEQIDNO:32, SEQIDNO:34, SEQIDNO:36, SEQIDNO:38, SEQIDNO:40, SEQIDNO:42, SEQIDNO:44 and SEQIDNO:46 has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the sequence identity of 99% or 100%.In some embodiments, CYP153A-reductase hybrid fusion polypeptide variants is derived from CYP153A (G307A) polypeptide of the extra large bacillus of water oil, wherein glycocoll (G) is substituted by alanine (A), and with derive from the P450RhF reductase domain fusion of bacterial classification NCIMB9784 of Rhod.Cytochrome P450 RhF is self-centered, and the substrate showing height is promiscuity, and catalysis functional group widely.In other embodiments, for implementing CYP153A-reductase hybrid fusion polypeptide variants disclosed by the invention and SEQIDNO:6, SEQIDNO:8, SEQIDNO:10, SEQIDNO:12, SEQIDNO:14, SEQIDNO:16, SEQIDNO:18, SEQIDNO:20, SEQIDNO:22, SEQIDNO:24, SEQIDNO:26, SEQIDNO:28, SEQIDNO:30, SEQIDNO:32, SEQIDNO:34, SEQIDNO:36, SEQIDNO:38, SEQIDNO:40, SEQIDNO:42, SEQIDNO:44 or SEQIDNO:46 has at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the sequence identity of 98% or at least 99%, and one or more replacement can be comprised, this can obtain useful feature of the present invention and/or character.In other embodiments, for implementing CYP153A-reductase hybrid fusion polypeptide variants disclosed by the invention and SEQIDNO:8, SEQIDNO:10, SEQIDNO:12, SEQIDNO:14, SEQIDNO:16, SEQIDNO:18, SEQIDNO:20, SEQIDNO:22, SEQIDNO:24, SEQIDNO:26, SEQIDNO:28, SEQIDNO:30, SEQIDNO:32, SEQIDNO:34, SEQIDNO:36, SEQIDNO:38, SEQIDNO:40, SEQIDNO:42, SEQIDNO:44 or SEQIDNO:46 has 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, the sequence identity of 91% or 90%.In other embodiments, the P450 catalyst structure domain of CYP153A-reductase hybrid fusion polypeptide variants is derived from the species except the extra large bacillus of water oil.These type of other species include but not limited to the bacterial classification of acinetobacter, Mycobacterium marinum, Polaromonassp., Bo Ku island alkane eating bacteria, fungi burkholderia (Burkholderiafungorum), crescent handle bacillus (Caulobactercrescentus), Hyphomonasneptunium, Rhodopseudomonas palustris (Rhodopseudomonaspalustris), the bacterial classification of Sphingomonas, the bacterial classification of mycobacterium.In other embodiments, the reductase domain of CYP153A-reductase hybrid fusion polypeptide variants is derived from the species except the bacterial classification of Rhod.These type of other species include but not limited to that Rhodococcus equi, radioresistance acinetobacter calcoaceticus, Burkholderia mallei, Burkholderia mallei, Ralstonia eutropha, resistance to metal covet copper bacterium.
In relevant embodiment, the present invention openly comprises CYP153A-reductase hybrid fusion polynucleotide variant, itself and SEQIDNO:5, SEQIDNO:7, SEQIDNO:9, SEQIDNO:11, SEQIDNO:13, SEQIDNO:15, SEQIDNO:17, SEQIDNO:19, SEQIDNO:21, SEQIDNO:23, SEQIDNO:25, SEQIDNO:27, SEQIDNO:29, SEQIDNO:31, SEQIDNO:33, SEQIDNO:35, SEQIDNO:37, SEQIDNO:39, SEQIDNO:41, SEQIDNO:43, SEQIDNO:45 or SEQIDNO:47 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the sequence identity of 98% or at least 99%.In some embodiments, nucleic acid sequence encoding has the CYP153A-reductase hybrid fusion polypeptide variants of one or more replacement, and this can obtain feature and/or the character of improvement of the present invention.In the embodiment that another is relevant, for implementing CYP153A-reductase hybrid fusion polypeptide variants disclosed by the invention by nucleotide sequence coded as follows, this nucleotide sequence and SEQIDNO:7, SEQIDNO:9, SEQIDNO:11, SEQIDNO:13, SEQIDNO:15, SEQIDNO:17, SEQIDNO:19, SEQIDNO:21, SEQIDNO:23, SEQIDNO:25, SEQIDNO:27, SEQIDNO:29, SEQIDNO:31, SEQIDNO:33, SEQIDNO:35, SEQIDNO:37, SEQIDNO:39, SEQIDNO:41, SEQIDNO:43, SEQIDNO:45 or SEQIDNO:47 has 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, the sequence identity of 91% or 90%.In one aspect of the method, the present invention openly relates to the CYP153A-reductase hybrid fusion polypeptide variants covered by the amino acid sequence of following nucleic acid sequence encoding, described nucleotide sequence under high stringency conditions with SEQIDNO:7, SEQIDNO:9, SEQIDNO:11, SEQIDNO:13, SEQIDNO:15, SEQIDNO:17, SEQIDNO:19, SEQIDNO:21, SEQIDNO:23, SEQIDNO:25, SEQIDNO:27, SEQIDNO:29, SEQIDNO:31, SEQIDNO:33, SEQIDNO:35, SEQIDNO:37, SEQIDNO:39, SEQIDNO:41, SEQIDNO:43, the nucleotide sequence of the corresponding basic total length of SEQIDNO:45 or SEQIDNO:47 is hybridized.In some embodiments, CYP153A-reductase hybrid fusion polypeptide variants is derived from the extra large bacillus species of water oil.In other embodiments, P450 hybrid fusion polypeptide is derived from the bacterial classification of acinetobacter, Mycobacterium marinum, Polaromonassp., Bo Ku island alkane eating bacteria, fungi burkholderia (Burkholderiafungorum), crescent handle bacillus (Caulobactercrescentus), Hyphomonasneptunium, Rhodopseudomonas palustris (Rhodopseudomonaspalustris), the bacterial classification of Sphingomonas, the bacterial classification of mycobacterium.
Change and sudden change
The variant polypeptide that the present invention uses refers to such polypeptide, and it has and wild type or at least one different amino acid whose amino acid sequence of template peptide.Such as variant (such as mutant) can have the one or more of following conserved amino acid replacement, include but not limited to aliphatic amino acid (such as alanine, valine, leucine and isoleucine) replaced by another aliphatic amino acid; Serine is replaced by threonine; Threonine is replaced by serine; Acidic residues (such as aspartic acid and glutamic acid) is replaced by another acidic residues; Residue (such as asparagine and glutamine) with amide group is replaced by another residue with amide group; Alkaline residue (such as lysine and arginine) and another alkaline residue exchange; And aromatic residue (such as phenylalanine and tyrosine) is replaced by another aromatic residue.In some embodiments, variant polypeptide have about 1,2,3,4,5,6,7,8,9,10,15,20,30,40,50,60,70,80,90,100 or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, add, insert or delete.Some the preferred fragments playing the polypeptide of variant or mutant function remain the some or all of biological functions (such as enzymatic activity) of corresponding wild type peptide.In some embodiments, described fragment remains at least 75%, at least 80%, at least 90%, at least 95%, at least 98% or higher biological function of corresponding wild type peptide.In other embodiments, fragment or mutant remain the biological function of about 100% of corresponding wild type peptide.In other embodiments, some fragments show the biological function increased than corresponding wild type peptide.Which measure amino acid residue can be substituted, insert or delete and the guide not affecting biologic activity can use computer program well known in the art to find, such as LASERGENE software (DNASTAR, Inc., Madison, WI).In some embodiments, fragment shows the biological function increased than corresponding wild type peptide or template peptide.Such as, fragment can show the enzymatic activity than corresponding wild type peptide or template peptide improvement at least 10%, at least 25%, at least 50%, at least 75% or at least 90%.In other embodiments, described fragment shows the enzymatic activity than corresponding wild type peptide or template peptide improvement at least 100%, at least 200% or at least 500%.
It should be understood that polypeptide of the present invention can have the Additional conservative not affecting in fact polypeptide function or nonessential 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.As known in the art (see Bowieetal. (1990) Science, 247:1306-1310), specific replacement can be measured whether be allowed to (that is, required biological function can not adversely be affected, such as the enzymatic activity of ω-hydroxylase).Conservative 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor for wherein amino acid residue had the amino acid residue of similar side chain the replacement that replaces.This area has defined the family of the amino acid residue with similar side chain.These families comprise amino acid (the such as lysine with basic side chain, arginine, histidine), there is amino acid (the such as aspartic acid of acid side-chain, glutamic acid), there is amino acid (the such as glycocoll of uncharged polar side chain, N, glutamine, serine, threonine, tyrosine, halfcystine), there is amino acid (the such as alanine of non-polar sidechain, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophane), there is amino acid (the such as threonine of the side chain of β-branching, valine, isoleucine), and there is amino acid (the such as tyrosine of beta-branched side, phenylalanine, tryptophane, histidine).
Variant can natural formation or create in vitro.Specifically, technique for gene engineering can be used to create this type of variant, such as direct mutagenesis, random chemical mutagenesis, the delete procedure of exonuclease III or the clone technology of standard.Alternatively, chemosynthesis or modification can be used to create this type of variant, mutant, fragment, analog or derivant.The method preparing variant is well known in the art.Such as can prepare variant by using Stochastic sum direct mutagenesis.Stochastic sum direct mutagenesis is (for example, see Arnold (1993) Curr.Opin.Biotech.4:450-455) well known in the art.Fallibility PCR can be used to obtain random mutagenesis (for example, see Leungetal. (1989) Technique1:11-15; AndCaldwelletal. (1992) PCRMethodsApplic.2:28-33).In fallibility PCR, actual PCR implements under the following conditions, and wherein the fidelity of repair of archaeal dna polymerase is lower, makes in the point mutation obtaining height ratio in the total length of PCR primer.In brief, in such processing, by nucleic acid (polynucleotide sequence of such as encode P450 protein or P450 hybrid fusion polypeptide) to be mutagenic and PCR primer, reaction buffer, MgCl 2, MnCl 2, Taq polymerase and suitable concn dNTP mixing, in the point mutation obtaining height ratio in the total length of PCR primer.Such as can use the nucleic acid that 20fmoles is to be mutagenic, the various PCR primer of 30pmole, reaction buffer (comprises 50mMKCl, 10mMTrisHCl (pH8.3), 0.01% gel, 7mMMgCl 2, 0.5mMMnCl 2, the Taq polymerase of 5 units, 0.2mMdGTP, 0.2mMdATP, 1mMdCTP and 1mMdTTP) and implement reaction.PCR can implement 30 circulations: 94 DEG C, 1min; 45 DEG C, 1min; And 72 DEG C, 1min.But those skilled in the art it should be understood that these parameters can suitably change.Then, by the nucleic acid clone of mutagenesis in suitable carrier, and evaluate by the activity of the polypeptide of the encoded by nucleic acid of mutagenesis.Oligonucleotide-directed mutagenesis can be used to implement direct mutagenesis, thus in the DNA of paid close attention to any clone, generate the point specific sudden change in position.Oligonucleotides mutagenesis has described (for example, see Reidhaar-Olsonetal. (1988) Science241:53-57) in the art.In brief, in such processing, be synthesized with the multiple double chain oligonucleotides in the DNA to cloning to be introduced of one or more sudden change, and be inserted into (polynucleotide sequence of such as encode P450 polypeptide or P450 hybrid fusion polypeptide) in the DNA of clone to be mutagenic.Reclaim the clone comprising the DNA of mutagenesis, and assess the activity of the polypeptide coded by them.
For generate the another kind of method of variant be assembling PCR.Assembling PCR relates to and is assembled into PCR primer by the potpourri of little DNA fragmentation.A large amount of different PCR reactions is parallel in the vial to be carried out, and the product of one of them reaction has caused the product (see United States Patent (USP) 5,965,408) of another reaction.The another kind of method generating variant is sexual PCR mutagenesis.In sexual PCR mutagenesis, but the random fragment of DNA molecular is in homologous recombination that is different that be forced between the DNA molecular of the external DNA sequence dna of height correlation based on the result of sequence homology.Then, in PCR reaction, overlapping (crossover) is made to be fixed by the extension of primer.Sexual PCR mutagenesis has described (for example, see Stemmer (1994) Proc.Natl.Acad.Sci.U.S.A.91:10747-10751) in publication known in the art.In addition, variant can also be created by mutagenesis in vivo.In some embodiments, in nucleotide sequence, generate random mutation by the sequence in bacterial isolates (such as coli strain) described in amplification, described bacterial strain carries sudden change in one or more DNA reparation approach.This type of mutator gene bacterial strain has higher random mutation rate than wild-type strain.In a kind of bacterial strain in these bacterial strains, DNA amplification sequence (polynucleotide sequence of P450 hybrid fusion polypeptide of such as encoding) finally will generate random mutation in DNA.The mutator gene bacterial strain being applicable to mutagenesis in vivo has described (for example, see International Patent Application Publication No.WO1991/016427) in the publication of this area.In addition, cassette mutagenesis can also be used to generate variant.In cassette mutagenesis, the region of a section little of double chain DNA molecule is different from the synthetic oligonucleotide box replacement of native sequences.Oligonucleotides comprises randomized native sequences that is whole and/or part usually.In addition, recursive ensemble mutagenesis (Recursiveensemblemutagenesis) still can be used to generate variant.Recursive ensemble mutagenesis is the algorithm for protein transformation (that is, protein mutagenesis), researches and develops the different groups of the mutant that this algorithm is correlated with for the production of phenotype, and the amino acid sequence of the member of these colonies is different.The method uses feedback mechanism to control continuously the cassette mutagenesis (for example, see Arkinetal. (1992) Proc.Natl.Acad.Sci., U.S.A.89:7811-7815) of several combination taken turns.In some embodiments, variant uses Exponential ensemble mutagenesis (exponentialensemblemutagenesis) to create.Exponential ensemble mutagenesis be use the uniqueness of high percent and functional variant to generate the method for combinatorial libraries, the wherein residue of several group randomization abreast, thus identify each change site, form the amino acid (for example, see Delegraveetal. (1993) Biotech.Res.11:1548-1552) of functional protein.In some embodiments, Reorganization is used to create variant, wherein encode in the nucleic acid of different polypeptide a large amount of, a part of nucleic acid is wherein merged, thus the chimeric nucleic acid sequence of establishment encoding chimera polypeptide is (such as in U.S. Patent No. 5,965,408 and 5,939, described in 250).
Expression vector
In some embodiments, by the mode of recombinant vector, polynucleotide (or gene) sequence is supplied to host cell, wherein said recombinant vector comprises the promoter be operably connected with polynucleotide sequence.In certain embodiments, promoter be Growth adjustment, organelle-specificity, the promoter of tissue-specific, induction type, formation type or cell-specific.In some embodiments, recombinant vector comprises and is selected from least one following sequence: the expression control sequenc operationally puted together with polynucleotide sequence; The selected marker of operationally puting together with polynucleotide sequence; The flag sequence operationally puted together with polynucleotide sequence; The purification part operationally puted together with polynucleotide sequence; The secretion sequence operationally puted together with polynucleotide sequence; And the target sequence of operationally to put together with polynucleotide sequence.Expression vector of the present invention comprises the polynucleotide sequence being applicable to the form expressing polynucleotide sequence in host cell.Those skilled in the art will be appreciated that the design of expression vector can depend on the factor of the selection of host cell such as to be transformed, the expression of required polypeptide and so on.Expression vector of the present invention be directed in host cell, thus the polypeptide that production polynucleotide sequence (as mentioned above) is by mentioned earlier encoded, comprise fused polypeptide.In prokaryotes (such as Escherichia coli), the gene major part expressing coded polypeptide uses the carrier comprising formation type or inducible promoter to implement, and wherein said promoter instructs the expression of fusion or non-fused polypeptide.The amino acid of some joins in the polypeptide of wherein encoding by fusion vector, usually joins amino terminal or the carboxyl terminal of recombinant polypeptide.This fusion vector usually play in following 3 kinds of objects one or more: the expression increasing recombinant polypeptide; Increase the dissolubility of recombinant polypeptide; And the purifying of recombinant polypeptide is contributed to by the effect playing part in affinity purification.Usually, in the carrier of amalgamation and expression, introduce proteoclastic cleavage site in fusion part and the junction of recombinant polypeptide.This makes after fusion polypeptide purification, and recombinant polypeptide is separated with fusion part.The example of the qualification sequence of the same race of this type of and they comprises factor Xa, fibrin ferment and enterokinase.The example of fusion expression vector comprises pGEX carrier (PharmaciaBiotech, Inc., Piscataway, NJ; Smithetal. (1988) Gene67:31-40), pMAL carrier (NewEnglandBiolabs, Beverly, and pRITS carrier (PharmaciaBiotech MA), Inc., Piscataway, N.J.), they are respectively by glutathione S-transferase (GST), and maltose E binding protein or a-protein are fused in target recombinant peptide respectively.
The example of inducible non-fusion E. coli expression vector comprises pTrc carrier (Amannetal. (1988) Gene69:301-315) and pET11d carrier (Studieretal., GeneExpressionTechnology:MethodsinEnzymology185, AcademicPress, SanDiego, Calif. (1990) 60-89).By pTrc carrier, target gene is expressed and depend on transcribing of the host RNA polymerase caused by the trp-lac promoter, fusion of hybridizing.By pET11d carrier, target gene is expressed and depend on transcribing of being caused by T7gn10-lac promoter, fusion, wherein said promoter is mediated by the viral rna polymerase of coexpression (T7gnl).This varial polymerases is by host strain (such as BL21 (DE3) and HMS174 (DE3)), and by resident's bacteriophage lambda of taking T7gn1 gene in, the getting off of control that transcribe in lacUV5 promoter supplies.Well known in the art (for example, see Sambrooketal. (1989) MolecularCloning:ALaboratoryManual for protokaryon and eukaryotic suitable expression system, secondedition, ColdSpringHarborLaboratory).The example of inducible non-fusion E. coli expression vector comprises pTrc carrier (Amannetal. (1988) Gene69:301-315) and PET11d carrier (Studieretal. (1990) GeneExpressionTechnology:MethodsinEnzymology185, AcademicPress, SanDiego, CA, pp.60-89).In certain embodiments, polynucleotide sequence disclosed by the invention is operably connected with the promoter derived by Phage T5.In one embodiment, host cell is yeast cells.In such an implementation, expression vector is Yeast expression carrier.Can pass through for by external nucleic acid (such as DNA), carrier is incorporated in protokaryon and eukaryotic by the multiple art-recognized technology be incorporated in host cell.For to transform or the suitable method of transfection host cell can find in such as Sambrooketal. (as mentioned above).With regard to stably transforming bacterial cells, known (depending on used expression vector and transformation technology) sub-fraction cell accepts and copy expression vector.In order to identify and select these transformants, the gene of encoding selectable markers (such as antibiotic resistance) can be incorporated in host cell together with paid close attention to gene.Selectable markers comprises those that give medicine resistance, such as but not limited to ampicillin, kanamycins, chloromycetin or tetracycline.The nucleic acid of encoding selectable markers can be incorporated in host cell with on the identical carrier of coding polypeptide of the present invention, or can be incorporated in host cell on the carrier separated.
Optional approach transformation
Host cell disclosed by the invention or microbial organisms comprise host strain or host cell, these host strains or host cell comprise some and change through genetic modification or modification, thus check specific sudden change to effect of enzymatic activity (that is, recombinant cell or microbial organisms).According to the pattern of the native enzyme approach existed in original host cell, can alternately use multiple optional genetic manipulation and change by a kind of host to another kind of host.In one embodiment, host strain can be used check the expression of CYP153A-reductase hybrid fusion polypeptide or its variant combined with other biological improvement on synthesis (such as enzyme).Host strain can contain a large amount of gene alterations, thus checks specific variable, and it includes but not limited to condition of culture, comprises fermented ingredient, carbon source (such as feed), temperature, pressure, the culture pollutional condition of attenuating and the level of oxygen.
In one embodiment, host strain contains the deletion of optional fadE and fhuA.Acyl-CoA dehydrogenase (FadE) is the enzyme wanted of overstating for fat metabolism acid.Second step in its catalyze fatty acid-utilising (beta oxidation), it is method long-chain fatty acid (acyl group-CoA) being fragmented into acetyl-CoA molecule.More specifically, in bacterium, the second step of the beta oxidation circulation of Fatty acid degradation is that acyl group-CoA is oxidized to 2-enol-CoA, and this is by FadE catalysis.When Escherichia coli lack FadE, it can not grow as in carbon source at fatty acid, but can grow on acetic acid esters.The fatty acid of any chain length can not be utilized to meet the phenotype reported to fadE bacterial strain, that is, the fadE mutants which had that wherein FadE function is destroyed.FadE gene can optionally knock out or weaken, and to guarantee can to accumulate in cell as the acyl group-CoA of the intermediate of derivative of fatty acid approach, acyl group-CoA all like this can change into derivative of fatty acid effectively.But; due at use sugar as under the condition of carbon source, the expression of FadE may be prevented, and therefore FadE can only to exist and can not effectively with for the ester synthase of acyl group-CoA substrate or other enzymes compete on a small quantity; so when using sugar as carbon source, it is optionally that fadE weakens.FadE is prevented due to catabolite repression.Escherichia coli and other preferred consumption sugars of microorganism many exceed fatty acid, like this when 2 kinds of sources all can obtain, son and first consumption sugar (see D.Clark, JBacteriol. (1981) 148 (2): 521-6) is regulated by preventing fad.In addition, lack sugar and there is the expression that fatty acid can induce FadE.Because fad regulates son (the comprising FadE) protein receptor of expressing to raising, and can effectively capture acyl group-CoA, so acyl group-CoA intermediate can be lost at beta-oxidation approach.Therefore, fadE gene is advantageously made to be knocked or to weaken.Because most of carbon source mainly glycosyl, optionally weakens FadE.Gene fhuA coding TonA protein, it is the transport Summing Factor acceptor (V.Braun (2009) JBacteriol.191 (11): 3431 – 3436) of the energy coupling in colibacillary adventitia.Its deletion is optional.FhuA deletes and makes the attack of cell to bacteriophage have more resistance, and this is favourable under some fermentation condition.Therefore, it is desirable to delete the fhuA in host cell, wherein said cell may experienced by potential pollution in fermentation runs.
In another embodiment, host strain (as mentioned above) also covers the optional process LAN of one or more genes following: fadR, fabA, fabD, fabG, fabH, fabV, and/or fabF.The example of this genoid is for deriving from colibacillary fadR, derive from the fabA of salmonella typhimurium (Salmonellatyphimurium) (NP_460041), derive from the fabD of salmonella typhimurium (NP_460164), derive from the fabG of salmonella typhimurium (NP_460165), derive from the fabH of salmonella typhimurium (NP_460163), derive from the fabV of comma bacillus (YP_001217283), and derive from the fabF of clostridium acetobutylicum (Clostridiumacetobutylicum) (NP_350156).One or more the process LAN of these genes (the biosynthetic enzyme of its encode fatty acid and regulatory factor) can increase tiring of fatty acid derived compounds under multiple condition of culture, comprises ω-OH fatty acid and derivant thereof.
In another embodiment, use coli strain as the host cell for the production of ω-OH fatty acid and derivant thereof.Similarly, (namely these host cells provide one or more biosynthesis genes, the gene of the biosynthetic enzyme of encode fatty acid and regulatory factor) optional process LAN, under multiple condition of culture, can increase or strengthen fatty acid derived compounds such as derivative of fatty acid (such as ω-OH fatty acid and α further like this, ω-diacid etc.) tire, wherein said gene includes but not limited to fadR, fabA, fabD, fabG, fabH, fabV and/or fabF.The example of gene alteration comprise derive from colibacillary fadR, the derive from salmonella typhimurium fabA of (NP_460041), derive from the fabD of salmonella typhimurium (NP_460164), derive from the fabG of salmonella typhimurium (NP_460165), derive from the fabH of salmonella typhimurium (NP_460163), derive from the fabV of comma bacillus (YP_001217283), and derive from the fabF of clostridium acetobutylicum (NP_350156).In some embodiments, the operon for synthesizing carrying these biosynthesis genes can be transformed and at cells, thus is able to the production checking the expression of P450 and/or strengthen ω-OH fatty acid and α, ω-diacid further under multiple condition of culture.This type of operon for synthesizing comprises one or more biosynthesis genes.Such as ifab138 operon is the operon of transformation, it comprises optional fatty acid biosynthesis genes, comprise the fabV deriving from comma bacillus, derive from the fabH of salmonella typhimurium, derive from the fabD of salmonella typhimurium, derive from the fabG of salmonella typhimurium, derive from the fabA of salmonella typhimurium, and/or deriving from the fabF of clostridium acetobutylicum, these genes may be used for the process LAN promoting derivative of fatty acid, to check specific condition of culture.A benefit of this type of operon for synthesizing is that the speed that ω-OH derivative of fatty acid is produced can be increased or strengthen further.
In some embodiments, for expressing ACP and biosynthetic enzyme (such as ω-hydroxylase, thioesterase etc.) host cell or microbial organisms express the gene containing some enzymatic activity further, wherein said enzymatic activity can increase the production of one or more special fatty acid derivants, such as ω-OH fatty acid, ω-OH derivative of fatty acid, α, ω-diacid etc.In one embodiment, described host cell has the thioesterase activity (E.C.3.1.2.*, E.C.3.1.2.14 or E.C.3.1.1.5) for the production of fatty acid, and wherein said thioesterase activity can be increased by the gene described in process LAN.In another embodiment, described host cell has the ester synthase activity (E.C.2.3.1.75) for the production of fatty ester.In another embodiment, described host cell has the active and/or alcohol dehydrogenase activity (E.C.1.1.1.1.) of acyl-acp reductase (AAR) (E.C.1.2.1.80) for the production of fatty alcohol and/or fatty alcohol acyl group-CoA reductase (FAR) (E.C.1.1.1.*) is active and/or carboxylate reductase (CAR) (EC1.2.99.6) is active.In another embodiment, acyl-acp reductase (AAR) (E.C.1.2.1.80) that described host cell has for the production of fatty aldehyde is active.In another embodiment, described host cell has active and decarboxylase (ADC) activity of acyl-acp reductase (AAR) (E.C.1.2.1.80) for the production of alkane and alkene.In another embodiment; acyl group-CoA the reductase (E.C.1.2.1.50) that described host cell has for the production of fatty alcohol is active, and acyl-CoA synthase (FadD) (E.C.2.3.1.86) is active and thioesterase (E.C.3.1.2.*, E.C.3.1.2.14 or E.C.3.1.1.5) is active.In another embodiment; described host cell has the ester synthase activity (E.C.2.3.1.75) for the production of fatty ester, and acyl-CoA synthase (FadD) (E.C.2.3.1.86) is active and thioesterase (E.C.3.1.2.*, E.C.3.1.2.14 or E.C.3.1.1.5) is active.In another embodiment, the OleA that described host cell has for the production of ketone is active.In another embodiment, the OleBCD that described host cell has for the production of internal olefin is active.In another embodiment, described host cell has the active and alcohol dehydrogenase activity (E.C.1.1.1.1.) of acyl-acp reductase (AAR) (E.C.1.2.1.80) for the production of fatty alcohol.In another embodiment, described host cell has the active and decarboxylase of thioesterase (E.C.3.1.2.*, E.C.3.1.2.14 or E.C.3.1.1.5) for the preparation of terminal olefin.The expression of enzymatic activity in microbial organisms and microbial cell by U.S. Patent number 8,097,439; 8,110,093; 8,110,670; 8,183,028; 8,268,599; 8,283,143; 8,232,924; 8,372,610 and 8,530,221 instructed, and these documents are incorporated herein by reference.In other embodiments, host cell or microbial organisms for expressing ACP and other biological synzyme comprise some natural enzymatic activity, these enzymes are raised or are over-expressed thus produced one or more specific derivative of fatty acid (multiple), such as ω-OH fatty acid, ω-OH derivative of fatty acid and α, ω-diacid.In one embodiment, the natural sulphur esterase (E.C.3.1.2.*, E.C.3.1.2.14 or E.C.3.1.1.5) that described host cell has for the production of fatty acid is active, and wherein said thioesterase can be increased by the thioesterase gene described in process LAN.
The present invention openly comprises host strain or microbial organisms, and it expresses the gene of coding CYP153A-reductase hybrid fusion polypeptide and variant and other biosynthetic enzyme (as mentioned above).Recombinant host cell produces derivative of fatty acid, and (such as ω-hydroxy fatty acid, comprises ω-hydroxyl free fatty acid to comprise ω-hydroxy fatty acid derivative; ω-hydroxy fatty acid methyl ester; ω-oxo fatty acid; ω-oxo fatty acid methyl ester; α, ω-diacid; α, omega-diol; α, ω-diester; ω-carboxylic fatty acids methyl ester; Omega-amino-fatty acid and omega-amino-fatty acid methyl ester), and their composition and blend.Derivative of fatty acid reclaims by culture medium usually, and/or is separated with host cell.In one embodiment, derivative of fatty acid reclaims by culture medium (extracellular).In another embodiment, derivative of fatty acid is reclaimed by host cell (in cell).In another embodiment, derivative of fatty acid is by reclaiming in culture medium and being separated with host cell.Methods known in the art (such as GC-FID) can be used to analyze the fatty acid derived compositions produced by host cell, thus (such as ω-hydroxy fatty acid, comprises ω-hydroxyl free fatty acid to measure the composition of the distribution of specific derivative of fatty acid and fatty acid derived compositions; ω-hydroxy fatty acid methyl ester; ω-oxo fatty acid; ω-oxo fatty acid methyl ester; Dual-function compound, such as α, ω-diacid; α, omega-diol; α, ω-diester; ω-carboxylic fatty acids methyl ester; Omega-amino-fatty acid; Omega-amino-fatty acid methyl ester etc.) chain length and saturation degree.
In other embodiments, host cell or microbial organisms for expressing ACP and other biosynthetic enzyme will comprise natural enzymatic activity, these enzymes are raised or are over-expressed, thus produce one or more specific derivative of fatty acid, (such as ω-hydroxy fatty acid, comprises ω-hydroxyl free fatty acid to comprise ω-hydroxy fatty acid derivative; ω-hydroxy fatty acid methyl ester; ω-oxo fatty acid; ω-oxo fatty acid methyl ester; Dual-function compound, such as α, ω-diacid; α, omega-diol; α, ω-diester; ω-carboxylic fatty acids methyl ester; Omega-amino-fatty acid and omega-amino-fatty acid methyl ester).In one embodiment, the natural sulphur esterase (E.C.3.1.2.*, E.C.3.1.2.14 or E.C.3.1.1.5) that described host cell has for the production of fatty acid is active, and wherein said thioesterase can be increased by process LAN thioesterase gene.
Recombinant host cell and fermentation
In order to produce ω-hydroxy fatty acid derivative, carry out a large amount of modifications, thus produce host cell (as mentioned above).Therefore, present disclosure provides recombinant host cell, its provide through transformation relative to do not transform or omega-3-fatty acids biosynthesis pathway for native host cell (such as playing the wild-type host cells of compared with control cells effect), this is completed by the improvement of such as specific bacterial strain.Production HOST ORGANISMS disclosed by the invention comprises plant, animal or microbial cell.The microbial organisms of such as bacterium, cyanobacteria, yeast, marine alga or filamentous fungi and so on or microbial cell can be used as to produce host.The non-limiting example that can be used as the microbial organisms producing host comprises Escherichia coli, saccharomyces cerevisiae etc. (as mentioned above).Microbial strains effectively changes into fatty acid or fatty acid ester, such as fatty acid methyl ester (FAME), fatty-acid ethyl ester (FAEE) and fatty alcohol (FALC) by from the glucose of reproducible feed or other carbon sources.In order to achieve the above object, described bacterial strain carefully transformed thus expresses crucial enzyme, comprising the thioesterase (such as deriving from colibacillary TesA) for the production of fatty acid or the ester synthase for the production of FAME (such as deriving from the ES9 except hydrocarbon sea bacillus).For the production of the scheme of the high density fermentation of multiple compounds and process set up (see U.S. Patent No. 8,372,610; 8,323,924; 8,313,934; 8,283,143; 8,268,599; 8,183,028; 8,110,670; 8,110,093 and 8,097,439, these documents are incorporated herein by reference).
The enzyme step (it plays the effect of biocatalyst) that technique for gene engineering can be used the present invention to be discussed adds in these microbial organisms, thus establishment can produce the difunctional derivative of fatty acid (α such as shown in Fig. 1 and 2, ω-diacid, and the α shown in Fig. 3 and 4, omega-diol) novel microbial organism.Product described in expectation is secreted into the outside of cell, thus allows by centrifugal and easily gather in the crops.Some recombinase step can be combined with a kind of microbial organisms, for directly producing specific compound, and such as, 12 amino dodecanoic acid shown in Fig. 1 and 2 or 12 amino dodecanoic acid methyl ester.Alternatively, use the biology of the intermediate produced by reproducible source to transform to may be used in fermentation process disclosed by the invention.The dodecylic acid methyl ester such as obtained by the recombinant microorganism organism of expressing enzyme (such as thioesterase or ester synthase) can be expressed enzyme (such as alcohol dehydrogenase or oxidase by infeed; Aminopherase or transaminase; And/or esterase or lipase) recombinant microorganism organism in.Like this, the chemical entities obtained by a kind of recombinant microorganism organism of some enzyme of expressing some step of catalysis can be fed in the another kind of recombinant microorganism organism of the enzyme of expressing other steps of catalysis.Therefore, described host cell provides production system, and wherein intermediate is tradable, and sweat may be used in all host cells, thus the ω-hydroxy fatty acid derivative needed for generating.It should be noted that by glucose or other reproducible feed (except Exogenous Fatty Acid or paraffin) directly and effectively produce ω-hydroxy fatty acid derivative (such as α, ω-diacid; α, omega-diol or omega-amino-fatty acid) method up to the present do not exist.But these difunctional derivative of fatty acid are important precursors for the synthesis of polymkeric substance.As proposed by the present invention for the production of the method based on fermentation of ω-hydroxy fatty acid derivative provide the chemical method that this area is used quick and eco-friendly alternative.
Present disclosure provides and (such as derive from the carbohydrates of corn, rattan or lignocellulose biomass by reproducible feed; Or the such as waste product of glycerine, waste gas, synthetic gas; Or the organic material improved, such as living beings) derivative carbon source directly produces ω-hydroxy fatty acid derivative.In one embodiment, described method depends on the effective and reproducible alternative source of cost for the production of these important chemical.Described method comprises produces ω-hydroxy fatty acid derivative by following process: in fermentation liquor, provide recombinant microorganism organism (such as host cell); Reproducible feed is added in fermentation liquor; And by separation of fermentative broth ω-hydroxy fatty acid derivative.The host cell of specific microbial organisms comprises the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises thioesterase or ester synthase and ω-hydroxylase.
As used herein, term fermentation refers to that organic material is converted into target substance by host cell in the broadest sense, such as by breeding the culture of recombinant host cell in the nutrient culture media comprising carbon source, the carbon source derived by reproducible feed is converted into ω-OH derivative of fatty acid and composition thereof by recombinant host cell.Allow the condition of producing to refer to and allow host cell to produce any condition of required product (such as ω-OH derivative of fatty acid).Similarly, one or more conditions that wherein polynucleotide of carrier are expressed refer to any condition allowing host cell improvement on synthesis.Suitable condition comprises such as fermentation condition.Fermentation condition can comprise such as many parameters, includes but not limited to that temperature range, ventilation level, feeding rate and nutrient culture media form.Each of these conditions (independent and combination) all allows host cell growth.Fermentation can be aerobic, anaerobism or their variant (such as microaerophilic).Exemplary culture medium comprises nutrient solution or gel.Usually, comprise can by the carbon source of the direct metabolism of host cell for nutrient culture media.In addition, in nutrient culture media, can enzyme be used, thus be conducive to the mobilization (such as starch or cellulose are depolymerized to fermentable sugar) of carbon source and metabolism subsequently.
With regard to small-scale production, the host cell through transformation can with batch growth of such as about 100 μ L, 200 μ L, 300 μ L, 400 μ L, 500 μ L, 1mL, 5mL, 10mL, 15mL, 25mL, 50mL, 75mL, 100mL, 500mL, 1L, 2L, 5L or 10L; Fermentation; And be induced and express required polynucleotide sequence, the CYP153A-reductase hybrid fusion polypeptide of such as encoding independent or the polynucleotide sequence with other enzyme functional combination.With regard to producing on a large scale, through transformation host cell can with such as approximately 10L, 100L, 1000L, 10,000L, batch growth of 100,000L, 1,000,000L or larger; Fermentation; And be induced and express required polynucleotide sequence.Alternatively, the fermentation of extensive batch feed can be implemented.ω-OH fatty acid derived compositions of the present invention can find in the extracellular environment of recombinant host cell culture, and can easily be separated by culture medium.ω-OH fatty acid or derivatives thereof can be secreted by recombinant host cell, be transported in extracellular environment or active transport in the extracellular environment of recombinant host cell culture.Conventional method known in the art is used to be separated its ω-OH derivative of fatty acid by recombinant host cell.
In some embodiments, host cell is cultivated in the culture medium comprising the carbon source of initial concentration (such as the reproducible feed of about 2g/L to about 100g/L).In other embodiments, culture medium comprises the carbon source that initial concentration is about 2g/L to about 10g/L, the carbon source of about 10g/L to about 20g/L, the carbon source of about 20g/L to about 30g/L, the carbon source of about 30g/L to about 40g/L or the carbon source of about 40g/L to about 50g/L.In some embodiments, fermentation situation is monitored for the level of carbon source in culture medium.In some embodiments, described method comprises further when the carbon source in nutrient culture media is lower than about 0.5g/L, in this culture medium, add supplementary carbon source.In some embodiments, when the level of carbon source in nutrient culture media is lower than about 0.4g/L, lower than about 0.3g/L, during lower than about 0.2g/L or lower than about 0.1g/L, supplementary carbon source is added in culture medium.In some embodiments, add supplementary carbon source, thus keep the level of carbon source to be about 1g/L to about 25g/L.In some embodiments, add supplementary carbon source, thus keep the level of carbon source to be about 2g/L or higher (such as about 2g/L or higher, about 3g/L or higher, about 4g/L or higher).In certain embodiments, add supplementary carbon source, thus keep the level of carbon source to be about 5g/L or lower (such as about 5g/L or lower, about 4g/L or lower, about 3g/L or lower).In some embodiments, add supplementary carbon source, thus keep the level of carbon source to be that about 2g/L is to about 5g/L, about 5g/L to about 10g/L or about 10g/L to about 25g/L.In some embodiments, carbon source is the reproducible feed of glucose or another kind of type, such as glycerine.
In some embodiments, production concentration is the ω-hydroxy fatty acid derivative of about 1g/L to about 200g/L.In some embodiments, production concentration is the ω-hydroxy fatty acid derivative of about 1g/L or higher (such as about 1g/L or higher, about 10g/L or higher, about 20g/L or higher, about 50g/L or higher, about 100g/L or higher).In some embodiments, production concentration is about 1g/L to the ω-hydroxy fatty acid derivative of about 170g/L, approximately 1g/L to about 10g/L, approximately 40g/L to about 170g/L, approximately 100g/L to about 170g/L, approximately 10g/L to about 100g/L, approximately 1g/L to about 40g/L, approximately 40g/L to about 100g/L or about 1g/L to about 100g/L.
In some embodiments, production is tired as about 25mg/L, about 50mg/L, about 75mg/L, about 100mg/L, about 125mg/L, about 150mg/L, about 175mg/L, about 200mg/L, about 225mg/L, about 250mg/L, about 275mg/L, about 300mg/L, about 325mg/L, about 350mg/L, about 375mg/L, about 400mg/L, about 425mg/L, about 450mg/L, about 475mg/L, about 500mg/L, about 525mg/L, about 550mg/L, about 575mg/L, about 600mg/L, about 625mg/L, about 650mg/L, about 675mg/L, about 700mg/L, about 725mg/L, about 750mg/L, about 775mg/L, about 800mg/L, about 825mg/L, about 850mg/L, about 875mg/L, about 900mg/L, about 925mg/L, about 950mg/L, about 975mg/L, about 1000mg/L, about 1050mg/L, about 1075mg/L, about 1100mg/L, about 1125mg/L, about 1150mg/L, about 1175mg/L, about 1200mg/L, about 1225mg/L, about 1250mg/L, about 1275mg/L, about 1300mg/L, about 1325mg/L, about 1350mg/L, about 1375mg/L, about 1400mg/L, about 1425mg/L, about 1450mg/L, about 1475mg/L, about 1500mg/L, about 1525mg/L, about 1550mg/L, about 1575mg/L, about 1600mg/L, about 1625mg/L, about 1650mg/L, about 1675mg/L, about 1700mg/L, about 1725mg/L, about 1750mg/L, about 1775mg/L, about 1800mg/L, about 1825mg/L, about 1850mg/L, about 1875mg/L, about 1900mg/L, about 1925mg/L, about 1950mg/L, about 1975mg/L, about 2000mg/L (2g/L), 3g/L, 5g/L, 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L, 80g/L, 90g/L, ω-the hydroxy fatty acid derivative of 100g/L or above-mentioned any 2 value limited ranges.In other embodiments, produce and tire as higher than 100g/L, higher than 200g/L, higher than the omega-3-fatty acids derivant of 300g/L or higher (such as 500g/L, 700g/L, 1000g/L, 1200g/L, 1500g/L or 2000g/L).Preferably tiring as 5g/L to 200g/L, 10g/L to 150g/L, 20g/L to 120g/L of the ω-hydroxy fatty acid derivative produced by recombinant host cell according to method disclosed by the invention, and 30g/L to 100g/L.In one embodiment, tiring as about 1g/L to about 250g/L of the ω-hydroxy fatty acid derivative produced by recombinant host cell according to method disclosed by the invention be more specifically 90g/L extremely about 120g/L.Tire and refer to the combination of concrete omega-3-fatty acids derivant or the omega-3-fatty acids derivant produced by given recombinant host cell culture.
In other embodiments, the host cell producing ω-hydroxy fatty acid derivative according to method disclosed by the invention through transformation has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 40% or by above-mentioned any 2 value limited ranges productive rate.In other embodiments, produce productive rate be higher than 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher ω-hydroxy fatty acid derivative or derivant.Alternatively or in addition, productive rate is about 30% or lower, about 27% or lower, about 25% or lower or about 22% or lower.Therefore, described productive rate can be limited by above-mentioned any 2 end points.ω-the hydroxy fatty acid derivative such as produced by recombinant host cell according to method disclosed by the invention or the productive rate of derivant can be 5% to 15%, 10% to 25%, 10% to 22%, 15% to 27%, 18% to 22%, 20% to 28% or 20% to 30%.In particular embodiments, the ω-hydroxy fatty acid derivative produced by recombinant host cell or the productive rate of derivant are about 10% to about 40%.In another specific embodiment, the ω-hydroxy fatty acid derivative produced by recombinant host cell or the productive rate of derivant are about 25% to about 30%.Productive rate can refer to the combination of concrete ω-hydroxy fatty acid derivative or the ω-hydroxy fatty acid derivative produced by given recombinant host cell culture.In addition, productive rate also depends on used feed.
In some embodiments, ω-the hydroxy fatty acid derivative produced by recombinant host cell or the throughput rate of derivant are at least 100mg/L/ hour, at least 200mg/L/ hour, at least 300mg/L/ hour, at least 400mg/L/ hour, at least 500mg/L/ hour, at least 600mg/L/ hour, at least 700mg/L/ hour, at least 800mg/L/ hour, at least 900mg/L/ hour, at least 1000mg/L/ hour, at least 1100mg/L/ hour, at least 1200mg/L/ hour, at least 1300mg/L/ hour, at least 1400mg/L/ hour, at least 1500mg/L/ hour, at least 1600mg/L/ hour, at least 1700mg/L/ hour, at least 1800mg/L/ hour, at least 1900mg/L/ hour, at least 2000mg/L/ hour, at least 2100mg/L/ hour, at least 2200mg/L/ hour, at least 2300mg/L/ hour, at least 2400mg/L/ hour or at least 2500mg/L/ hour.ω-the hydroxy fatty acid derivative such as produced by recombinant host cell according to method disclosed by the invention or the throughput rate of multiple derivant can be little of 2500mg/L/ hour for 500mg/L/, or 700mg/L/ is little of 2000mg/L/ hour.In a specific embodiment, throughput rate is about 0.7mg/L/h to about 3g/L/h.Throughput rate can refer to the combination of concrete ω-hydroxy fatty acid derivative or the ω-hydroxy fatty acid derivative produced by given recombinant host cell culture.
The strategy producing ω-OH aliphatic acid composition for increasing recombinant host cell comprises the flow by fatty acid biosynthetic pathway increased by process LAN CYP153A-reductase hybrid fusion gene in type of production host and thioesterase gene.As used herein, term recombinant host cell or the host cell through transforming refer to such host cell, such as, by the new Genetic elements of premeditated introducing and/or the premeditated host cell naturally occurring Genetic elements that is modified at, gene formation is changed for corresponding wild-type host cells.In addition, the offspring of this type of recombinant host cell also comprises these new and/or modified Genetic elements.In either side in disclosure of the invention of the present invention, host cell can be selected from vegetable cell, insect cell, (such as filamentous fungi, as candida strain for fungal cell; Or budding yeast, such as barms), alginic cell and bacterial cell.In one embodiment, recombinant host cell is restructuring microbial organisms.Example as the host cell of microbial organisms includes but not limited to derive from Colibacter (Escherichia), bacillus (Bacillus), Lactobacillus (Lactobacillus), zymomonas (Zymomonas), Rhod (Rhodococcus), pseudomonas (Pseudomonas), Eurotium (Aspergillus), trichoderma (Trichoderma), neurospora (Neurospora), Fusarium (Fusarium), the mould genus of humic (Humicola), Rhizomucor (Rhizomucor), genus Kluyveromyces (Kluyveromyces), pichia (Pichia), mucor (Mucor), the erosion mould genus of silk (Myceliophtora), Penicillium (Penicillium), the flat lead fungi of raw wool belongs to (Phanerochaete), Pleurotus (Pleurotus), Trametes (Trametes), Chrysosporium (Chrysosporium), Blastocystis (Saccharomyces), Stenotrophomonas belongs to (Stenotrophamonas), Schizosaccharomyces (Schizosaccharomyces), the cell of Ye Shi saccharomyces (Yarrowia) or streptomyces (Streptomyces).In some embodiments, host cell is gram positive bacterial cell.In other embodiments, host cell is gram negative bacterial cell.In some embodiments, host cell is Bacillus coli cells.In some embodiments, host cell is Escherichia coli B cell, Escherichia coli C cell, Escherichia coli K cell or Escherichia coli W cell.In other embodiments, host cell is bacillus lentus (Bacilluslentus) cell, bacillus brevis (Bacillusbrevis) cell, bacillus stearothermophilus (Bacillusstearothermophilus) cell, bacillus licheniformis (Bacilluslichenoformis) cell, Alkaliphilic bacillus (Bacillusalkalophilus) cell, bacillus coagulans (Bacilluscoagulans) cell, Bacillus circulans (Bacilluscirculans) cell, bacillus pumilus (Bacilluspumilis) cell, bacillus thuringiensis (Bacillusthuringiensis) cell, Bacillus clausii (Bacillusclausii) cell, bacillus megaterium (Bacillusmegaterium) cell, bacillus subtilis (Bacillussubtilis) cell, or separate starch tooth born of the same parents bacillus (Bacillusamyloliquefaciens) cell.In other embodiments, host cell is koning trichoderma (Trichodermakoningii) cell, Trichoderma viride (Trichodermaviride) cell, trichoderma reesei (Trichodermareesei) cell, long stalk wood mould (Trichodermalongibrachiatum) cell, aspergillus awamori (Aspergillusawamori) cell, aspergillus fumigatus (Aspergillusfumigates) cell, smelly aspergillus (Aspergillusfoetidus) cell, aspergillus nidulans (Aspergillusnidulans) cell, aspergillus niger (Aspergillusniger) cell, aspergillus oryzae (Aspergillusoryzae) cell, Humicola insolens (Humicolainsolens) cell, cotton wool shape humicola lanuginosa (Humicolalanuginose) cell, opaque Rhodococcus cells, rhizomucor miehei (Rhizomucormiehei) cell or rice black wool mould (Mucormichei) cell.In another embodiment, host cell is muta lead mycillin (Streptomyceslividans) cell or grey streptomycete (Streptomycesmurinus) cell.In other embodiments, host cell is actinomyces (Actinomycetes) cell.In some embodiments, host cell is saccharomyces cerevisiae (Saccharomycescerevisiae) cell.
In other embodiments, host cell be eukaryotic plant cell, alginic cell, cyanobacteria cell, green sulphur bacteria cell, green non-sulfur bacteria cell, purple sulfur bacteria cell, purple nonsulfur bacteria cell, extremophile biological cell, yeast cells, fungal cell, any species of the present invention through transformation cell or synthesis organism.In some embodiments, host cell is that light relies on or fixed carbon.In some embodiments, host cell has autotrophy activity.In some embodiments, such as in the presence of light, host cell has photoautotrophy activity.In some embodiments, under light lacks, host cell is heterotrophism or holds concurrently foster.In certain embodiments, host cell is for deriving from arabidopsis (Arabidopsisthaliana), switchgrass (Panicumvirgatum), huge awns (Miscanthusgiganteus), corn (Zeamays), Wild Vitis species (Botryococcusebraunii), Chlamydomonas reinhardtii (Chlamydomonasreinhardtii), Dunaliella salina (Dunalielasalina), poly-Coccus bacterial classification PCC7002, poly-Coccus bacterial classification PCC7942, cytoalgae (SynechocystisSp.) bacterial classification PCC6803, long thermophilic poly-ball indigo plant (Thermosynechococcuselongates) BP-1, green sulphur bacteria (Chlorobiumtepidum), Chlorojlexusauranticus, Chromatiummvinosum, Rhodospirillum rubrum (Rhodospirillumrubrum), Rhodobacter capsulatus (Rhodobactercapsulatus), Rhodopseudomonas palustris (Rhodopseudomonaspalusris), Young clostridium (Clostridiumljungdahlii), Clostridium thermocellum (Clostridiumthermocellum), penicillium chrysogenum (Penicilliumchrysogenum), Pichia yeast (Pichiapastoris), saccharomyces cerevisiae, grain brewer yeast (Schizosaccharomycespombe), the cell of fluorescent pseudomonas (Pseudomonasfluorescens) or zymomonas mobilis (Zymomonasmobilis).In one embodiment, microbial cell derives from cyanobacteria, it includes but not limited to prochlorococcus (Prochlorococcus), Synechococcus (Synechococcus), cytoalgae (Synechocystis), blue bar algae (Cyanothece) and Nostocpunctiforme.In another embodiment, microbial cell derives from specific cyanobacteria species, and it includes but not limited to elongated Synechococcus (Synechococcuselongatus) PCC7942, the species PCC6803 of cytoalgae and the species PCC7001 of Synechococcus.
The product derived by recombinant host cell
As used herein, contemporary carbon level part or fM have the identical implication defined with NationalInstituteofStandardsandTechnology (NIST) StandardReferenceMaterialsSRMs4990B with 4990C (it is called oxalic acid standard items HOxI with HOxII).Basic definition refers to that 0.95 is multiplied by 14c/ 12c isotope ratio HOxI (see AD1950).This is roughly equivalent to the timber before the industrial revolution that decay correction crosses.For current living organism circle (vegetable material), fM is about 1.1.Biologic (such as derivative of fatty acid comprises the ω-OH fatty acid and derivant openly produced according to the present invention) comprises the organic compound that biological method is produced.Specifically, the derivative of fatty acid (such as ω-OH fatty acid and derivant thereof) using fatty acid biosynthetic pathway of the present invention to produce is not use renewable source to produce, and is therefore the composition of new material.Based on dual carbon isotope fingerprint method (dualcarbon-isotopicfingerprinting) or 14c determines year method and these new biologics and the organic compound being derived from petrochemistry carbon can be differentiated.In addition, the concrete source (such as glucose and glycerine) of bioresource carbon can be determined by dual carbon isotope fingerprint method (for example, see U.S. Patent No. 7,169,588).In business, the ability of difference biologic and petroleum base organic compound is useful following the trail of in these materials.Such as, the organic compound or the chemicals that comprise biologically-based and petroleum-based both carbon isotope spectrums can be different from the organic compound and chemicals be only made up of petroleum based material.Therefore, in business, can follow the trail of based on the carbon isotope spectrum of the uniqueness of biologic of the present invention or follow the tracks of them.By compare stable carbon isotope ratio in each sample ( 13c/ 12c), biologic and petroleum base organic compound can be distinguished.In given biologic 13c/ 12c is than being when in atmospheric carbon dioxide, this carbon dioxide is fixed 13c/ 12the result of C ratio.It also reflects metabolic pathway accurately.In addition, domain of the existence sex differernce is gone back.Oil, C3 plant (broad leaved plant), C4 plant (herbaceous plant) and marine carbon Barbiturates exist 13c/ 12c and corresponding δ 13all significant difference is shown in C value.Further, due to metabolic pathway, the analysis situation of the fat material of C3 with C4 plant is different from the material of the carbohydrate ingredient derived from identical plant.Within measuring accuracy, due to isotopic fractionation 13c shows large difference, and for biologic, the most significant effect is photosynthetic mechanism.In plant, in carbon isotope ratio, in the main cause of difference and plant, the difference of photosynthetic carbon metabolism approach is closely related, particularly in elementary carboxylation (that is, Atmospheric CO 2initial fixing) reaction that occurs of period.Two large class plants relate to the photosynthetic circulation of " C3 " (or Calvin-Benson) and relate to the plant of C4 (or Hatch-Slack) photosynthetic circulation.In C3 plant, main CO 2fixing or carboxylation reaction relates to ribulose-1,5-bisphosphate, 5-diphosphonic acid carboxylase, and the first stable product is 3-carbon compound.The C3 plant of such as broad leaf tree and conifer and so on is main in temperate climate area.In C4 plant, other carboxylation reaction relating to another kind of enzyme (phosphoric acid enol pyruvic acid carboxylase) is main carboxylation reaction.First stable carbon compound is the acid of 4-carbon, and the acid of this 4-carbon is subsequently by decarboxylation.The CO of release like this 2circulated by C3 and fix again.The example of C4 plant is tropical pasture, corn and sugarcane.C4 and C3 plant all show certain limit 13c/ 12c isotope ratio, but for C4 plant, common value is approximately-7 to about-13/ mil, and for C3 plant, approximately-19 to about-27/ mil (for example, see Stuiveretal, Radiocarbon19:355 (1977)).In the scope that coal and oil lag behind usually. 13c measurement scale defined by the null set of PeeDeeBelemnite (PDB) lime stone at first, and the value wherein provided is the permillage with the deviation of this material.δ 13C value is expressed as permillage (every mil), is abbreviated as ‰, and calculates according to the following formula:
δ 13c (‰)=[( 13c/ 12c) sample-( 13c/ 12c) standard items]/( 13c/ 12c) standard items x1000
Because PDB reference material (RM) is depleted, develop a series of alternative RM cooperatively with IAEA, USGS, NIST and other selected international isotopic laboratory.With the per unit mil deviation of PDB be labeled as δ 13c.By high-precision stable ratio mass spectrum (IRMS) on the molion of quality 44,45 and 46 to CO 2measure.Composition of the present invention comprises the biologic produced by any method of the present invention, comprises such as derivative of fatty acid product.Specifically, the δ of described biologic 13c can be approximately-28 or larger, approximately-27 or larger ,-20 or larger ,-18 or larger ,-15 or larger ,-13 or larger ,-10 or larger or-8 or larger.The δ of such as described biologic 13c can be about-30 to about-15, about-27 to about-19, about-25 to about-21, about-15 to about-5, about-13 to about-7 or about-13 to about-10.In other cases, the δ of described biologic 13c can be about-10 ,-11 ,-12 or-12.3.In addition, can also by more often kind of compound 14the amount of C, is different from petroleum base organic compound by according to the open biologic produced of the present invention.Because 14c has the core half life period of 5730, so the petroleum based fuels comprising older carbon can be different from comprise newer carbon biologic (see such as Currie, " SourceApportionmentofAtmosphericParticles ", CharacterizationofEnvironmentalParticles, J.BuffleandH.P.vanLeeuwen, Eds., 1ofVol.IoftheIUPACEnvironmentalAnalyticalChemistrySeries (LewisPublishers, Inc.) 3-74, (1992)).The basic assumption of radiocarbon dating method is in air 14the constancy of C concentration causes in Living Organism 14the constancy of C.But, due to the air nuclear test since nineteen fifty and the burning of fossil fuel since 1850, 14c has obtained secondary geochemistry temporal characteristics (geochemicaltimecharacteristic). 14c is in Atmospheric CO 2in and concentration thus in biosphere almost double when the peak of 19th century the mid-1960s nuclear test.After this, it is little by little back to about 1.2x10 -12stable state universe occur (air) baseline isotope rate ( 14c/ 12c), relaxation " half life period " (relaxation " half-life ") and is roughly 7 to 10 years.This half life period described in the latter need not according to literal understanding; But detailed air core input/decay function must have been used to the air since following the trail of the nuclear age and starting and biosphere 14the change of C.This biosphere described in the latter 14the annual Ding Nianfa of C temporal characteristics support measures the promise of the carbon in biosphere in modern age.Accelerator mass spectrometry (AMS) can be passed through measure 14c, result provides with the unit of the ratio of contemporary carbon (fM).FM is defined by NationalInstituteofStandardsandTechnology (NIST) StandardReferenceMaterials (SRM) 4990B and 4990C.As used herein, level part of contemporary carbon or fM have the identical implication defined with NationalInstituteofStandardsandTechnology (NIST) StandardReferenceMaterials (SRMs) 4990B with 4990C (it is called oxalic acid standard items HOxI with HOxII).Basic definition relates to 0.95 and is multiplied by 14c/ 12c isotope ratio HoxI (with reference to AD1950).This is roughly equivalent to the timber before the industrial revolution of decay correction.For current living organism circle (vegetable material), fM is about 1.1.Composition of the present invention comprises can fM 14c is the biologic of at least about 1.Such as biologic disclosed by the invention can have the fM of at least about 1.01 14the fM of C, about 1 to about 1.5 14the fM of C, about 1.04 to about 1.18 14the fM of C or about 1.111 to about 1.124 14c.
Known 14the another kind of C measures the percent (pMC) into contemporary carbon.For use 14the archaeologist in C age or sand smeller, AD1950 equals zero year.It also represents 100pMC.When the peak of thermonuclear weapon in 1963, the bomb carbon in air reaches the almost twice of normal level.Its distribution has in an atmosphere been approximate since it occurs, shows for the plant and animal of living since AD1950 the value being greater than 100pMC.As time goes by, this value reduces gradually, and current value is close to 107.5pMC.This means that the newborn substance material of such as corn and so on will provide close to 107.5pMC's 14c marks.Petroleum-based compounds by have be zero pMC value.The combination of fossil-carbon and contemporary carbon causes modern pMC content to be diluted.Modern biotechnology material is represented by supposition 107.5pMC 14c content and 0pMC represents petroleum-based products 14c content, then the measurement pMC value of this material is by the ratio of the composition of reflection two type.The radiocarbon C14 provided close to 107.5pMC marks by such as 100% material deriving from modern soybean.If use petroleum-based products by this material dilution 50%, then it will provide the radiocarbon C14 mark of about 54pMC.Equal 107.5pMC by specifying 100% and be appointed as 0% equal 0pMC and derivative to obtain based on biological carbon content.The sample being such as measured as 99pMC will provide the equivalent biologically-based carbon content of 93%.This value is called as based on biological average carbon result and supposes that all the components in analyzed material all originates from modern biomaterial or petroleum based material.The biologic comprising one or more derivative of fatty acid of the present invention can have at least about pMC of 50,60,70,75,80,85,90,95,96,97,98,99 or 100.In other cases, derivative of fatty acid of the present invention can have the pMC of about 50 to about 100, about 60 to about 100, about 70 to about 100, about 80 to about 100, about 85 to about 100, about 87 to about 98 or about 90 to about 95.In other cases, derivative of fatty acid of the present invention can have about pMC of 90,91,92,93,94 or 94.2.
ω-hydroxy fatty acid derivative and formulation
The compound of such as ω-hydroxy fatty acid derivative and so on is the molecule that tool is valuable and desirable in many commercial Application.The present invention is open produces this compounds in vivo by recombinant microorganism organism, generates multiple useful product thus.This type of product comprises ω-hydroxy fatty acid derivative.ω-hydroxy fatty acid derivative includes but not limited to ω-hydroxy fatty acid; ω-hydroxy-fatty acid methyl ester; Omega-amino-fatty acid; ω-oxo fatty acid, omega-amino-fatty acid methyl ester; ω-oxo fatty acid methyl ester; α, ω-diacid; α, ω-diester; α, omega-diol and their combination.Although the invention describes the ω-hydroxy-fatty acid derivative of the even number chain in large portion, also comprise the ω-hydroxy-fatty acid derivative of odd number chain, such as, there are 7,9,11,13,15,19 etc. carbon those.
The example of ω-hydroxy fatty acid includes but not limited to 8-Hydroxyoctanoic acid, 10-hydroxyl diluted acid in the last of the ten Heavenly stems, 10-hydroxydecanoic acid, 12-hydroxyl dodecenoic acid, sabinic acid, 14-hydroxyl tetradecenoic acid, 14-hydroxyl tetradecane acid, 16-hydroxy-16 carbon olefin(e) acid, juniperic acid, 18-hydroxyoctadecanoic acid and 18-kamalolenic acid.
The example of ω-hydroxy-fatty acid methyl ester includes but not limited to 8-Hydroxyoctanoic acid methyl ester, 10-hydroxyl diluted acid in last of the ten Heavenly stems methyl ester, 10-hydroxydecanoic acid methyl ester, 12-hydroxyl dodecenoic acid methyl ester, sabinic acid methyl ester, 14-hydroxyl tetradecenoic acid methyl ester, 14-hydroxyl tetradecane acid methyl ester, 16-hydroxy-16 carbon olefin(e) acid methyl ester, juniperic acid methyl ester, 18-hydroxyoctadecanoic acid methyl ester and 18-kamalolenic acid methyl ester.
The example of omega-amino-fatty acid includes but not limited to 8-aminocaprylic acid, 10-diluted acid in the amino last of the ten Heavenly stems, 10-aminocapric acid, the amino dodecenoic acid of 12-, 12 amino dodecanoic acid, the amino tetradecenoic acid of 14-, the amino tetradecanoic acid of 14-, the amino gaidic acid of 16-, 16-aminohexadecanoic acid, the amino octadecanoid acid of 18-and the amino 18 carbon diluted acids of 18-.
The example of omega-amino-fatty acid methyl ester includes but not limited to 8-aminocaprylic acid methyl ester, 10-diluted acid methyl ester in the amino last of the ten Heavenly stems, 10-aminocapric acid methyl ester, the amino dodecenoic acid methyl ester of 12-, 12 amino dodecanoic acid methyl ester, the amino tetradecenoic acid methyl ester of 14-, the amino tetradecanoic acid methyl ester of 14-, the amino gaidic acid methyl ester of 16-, 16-aminohexadecanoic acid methyl ester, the amino octadecanoid acid methyl ester of 18-and the amino 18 carbon diluted acid methyl ester of 18-.
The example of ω-oxo fatty acid includes but not limited to 8-oxo octanoic acid, 10-oxo diluted acid in the last of the ten Heavenly stems, 10-oxo capric acid, 12-oxo dodecenoic acid, 12-oxo dodecylic acid, 14-oxo tetradecenoic acid, 14-oxo tetradecanoic acid, 16-oxo gaidic acid, 16-oxo hexadecanoic acid, 18-oxo octadecanoid acid and 18-oxo 18 carbon diluted acid.
The example of ω-oxo fatty acid methyl ester includes but not limited to 8-oxo octanoic acid methyl ester, 10-oxo decylenic acid methyl ester, 10-oxo capric acid methyl ester, 12-oxo 12 carbon diluted acid methyl ester, 12-oxo lauric acid methyl ester, 14-oxo 14 carbon diluted acid methyl ester, 14-oxo tetradecanoic acid methyl ester, 16-oxo hexadecene acid methyl ester, 16-oxo hexadecanoic acid methyl ester, 18-oxo 18 carbon diluted acid methyl ester and 18-oxo 18 carbon diluted acid methyl ester.
The example of α, ω-diacid include but not limited to 1,8-sad, 1,10-decenedioic acid (decenedioicacid), 1,10-decanedioic acid, 1,12-decene dicarboxylic acid, 1,12-dodecanedioic acid, 1, the rare diacid of 14-14 carbon, 1,14-tetracosandioic acid, 1,16-hexadeeene diacid(hexadecendioic acid), 1,16-hexadecandioic acid (hexadecane diacid), 1,18-octadecene diacid, 1,18-octadecane diacid.
The example of α, ω-diester includes but not limited to the sad dimethyl esters of 1,8-, 1,10-decylenic acid dimethyl esters, 1,10-capric acid dimethyl esters, 1,12-dodecenoic acid dimethyl esters, 1,12-dodecylic acid dimethyl esters, 1,14-tetradecenoic acid dimethyl esters, 1,14-tetradecanoic acid dimethyl esters, 1,16-gaidic acid dimethyl esters, 1,16-hexadecanoic acid dimethyl esters, 1,18-octadecenoic acid dimethyl esters, 1,18-ten eight dimethyl carbonate.
α, the example of omega-diol includes but not limited to 1,8-ethohexadiol, 1,10-decene glycol, 1,10-decanediol, 1,12-dodecene glycol, 1,12-dodecanediol, 1,14-tetradecene glycol, 1,14-tetradecane diols, 1,16-hexadecylene glycol, 1,16-hexadecane diol, 1,18-octadecylene glycol and 1,18-octacosanol.
Although the invention describes the ω-hydroxy-fatty acid derivative of even number chain, also comprise the ω-hydroxy-fatty acid derivative of odd number chain, such as, there are 7,9,11,13,15,19 etc. carbon those.The example of odd number chain ω-hydroxy fatty acid includes but not limited to such as 11-hydroxyl 11 carbon diluted acid, 11-hydroxyl undecanoic acid, 13-hydroxyl 13 carbon diluted acid, 13-hydroxyl tridecylic acid, 15-hydroxyl 15 carbon diluted acid, 15-hydroxy-pentadecanoic acid, 17-hydroxyl 17 carbon diluted acid and 17-hydroxyl margaric acid.Odd number chain α, the example of ω-diacid includes but not limited to such as 1,11-nonene dicarboxylic acid, 1,11-dodecanedioic acid, 1,13-tridecylendioic acid, 13-tridecylic acid, 1,15-pentadecendioic acid, 1,15-pentadecanoic acid, 1,17-heptadecene diacid and 1,17-margaric acid.Odd number chain α, the example of omega-diol includes but not limited to such as 1,11-hendecene glycol, 1,11-undecane, 1,13-tridecylene glycol, 13-tridecane diols, 1,15-ten pentaene glycol, 1,15-pentadecane diols, 1,17-ten seven enediol and 1,17-heptadecane diols.
Compound and the composition of ω-hydroxy fatty acid derivative disclosed by the invention can be prepared, required product can be prepared like this, such as spices, essence, lubricant, gel, multiple polymers, resin, industrial fluids, bonding agent, corrosion inhibitor, capacitor electrolyte, fiber, powder coating medicine, plastifier, polyester coating, epoxy resin, polyamide, surfactant, washing agent, adjuvant etc.
Embodiment
Following examples further illustrate the present invention open, but should not be interpreted as defining scope of the present invention by any way.
Embodiment 1: cultivate the recombinant escherichia coli strain for the production of ω-hydroxy fatty acid derivative
Make described bacterial strain overnight growth on Luria-Bertani (LB) nutrient culture media.Described culture is diluted to fresh LB nutrient culture media with 1:10, at 32 DEG C, grows 2-3 hour (h), and then be diluted to the FA2 nutrient culture media (see table 10, vide infra) of definition with 1:10.In some embodiments, 0.5mM ALA and trace vitamin solution is used to supplement described nutrient culture media.When bacterial strain comprises the plasmid with various antibiotic-resistance marker, add the microbiotic of such as miramycin (100 μ g/ml) and kanamycins (50 μ g/ml) and so on.Grow 4-5h at 32 DEG C after, use 1mMIPTG Induced cultures and cultivate 16-18h more at that same temperature.Some test in, add dodecylic acid, dodecanol or dodecylic acid methyl ester, its induce time ultimate density be 1g/L.When inducing, the supplementary object of methyl alcohol (2%, v/v) is used to be the culture of the derivant producing fatty acid methyl ester.
Table 10:FA2 nutrient culture media composition
Medium component Ultimate density
5x M9 salt solusion 1x
100g/L NH4Cl 1g/L
10mg/mL thiamine 1μg/L
1M MgSO4 1mM
1M CaCl2 1mM
500g/L glucose 30g/L
1000x trace mineral solution (TM2) 1x
10g/L Fe citrate 10mg/L
2M BisTris(pH7.0) 100mM
10％Triton X-100 0.25％
The analysis of embodiment 2: ω-hydroxylated derivative of fatty acid
Results produce the culture of ω-hydroxy fatty acid derivative, and use vortice (DVX-2500 multitube vortice, VWR) to use butyl acetate to be extracted by culture in 30 minutes under 2500rpm.At room temperature, by extract in Eppendorf hydro-extractor (hydro-extractor 5424) under 15000rpm centrifugal 15 minutes.Being sucked by supernatant (100 μ L) has in the GC bottle of label, by adding 100 μ LN, two (trimethyl silyl) trifluoroacetamide (BSTFA) of O-and 1% trimethyl chlorosilane (TMCS) and derivatization, and use vortice to mix 30 seconds.The supernatant carrying out derivatization and do not carry out derivatization is injected on GC-MS, thus generates the chromatogram and mass spectrum that are used for compound identification.GC-MS parameter is as follows:
GC parameter:
Analytical column: DB-1HT, 15m × 250 μm × 0.1 μm, derive from Agilent, be numbered #J & W122-1111E
Furnace temperature: be initially 50 DEG C, keep 5 minutes, be increased to 300 DEG C, and keep 5.24 minutes with 25 DEG C/min, total runtime is 24 minutes
Column flow rate: 1.2mL/ minute
Temperature in: 300 DEG C
Sample size: 1 μ L
Split ratio: 20:1
Software: ChemStationE.02.01.1177
MS parameter
The temperature of transfer conduit: 300 DEG C
MS source: 230 DEG C
MSQuad：150℃
Self-actuated sampler
The CombiPAL (CTCanalytics) distributed by LEAPTechnologies
GC/FID parameter:
Be equipped with the GasChromatographAgilent7890 of FID or equivalent
Data system ChemStation software B.04.03, or equivalent
Analytical column: DB-1 (10m × 0.18mm × 0.2 μM), or equivalent
Self-actuated sampler: CombiPAL, CTCanalytics (LeapTechnologies)
Initial temperature: 60 DEG C, keep 0.5 minute, be increased to 300 DEG C, and keep 0.9 minute with 25 DEG C/min, total runtime is 11 minutes
Injector temperature: 320 DEG C
Detecting device: flame ionization detector (FID)
Detector temperature: 350 DEG C
Hydrogen flow rate: 40mL/ minute
Air velocity: 450mL/ minute
Constituent flow velocity: 45mL/ minute (N 2)
Split ratio: 50:1
Column flow rate: 0.8mL/ minute
Sample size: 1 μ L
Embodiment 3: dodecylic acid is changed into 12 (ω)-hydroxy-dodecanoic acids by the coli strain of expression 2 kinds of cyp153AP450 oxidase operons
The convert fatty acids that external source is added by the recombinant escherichia coli strain of expressing cyp153AP450 oxidase operon by the present embodiment display becomes ω-hydroxy fatty acid.The object of this test is the efficiency studying cyp153AP450 oxidase operon.Up to the present, cyp153AP450 oxidase only uses (for example, see Honda-Malcaetal. (2012) Chem.Commun.48:5115-5117) in vitro, and the object of this test detects when providing Exogenous Fatty Acid in vivo, and whether these operons can produce ω-hydroxy fatty acid derivative.
Derive from 2 kinds of bacterium (Marinobacteraquaeoli (numbering YP_957888; And Mycobacterium marinum (numbering YP_001851443 SEQIDNO:3); SEQIDNO:59) cyp153A operon) carries out pcr amplification by these organic genomic DNAs.Described operon is by the genomic constitution (see table 2A and 2B, described above) of encoding ferredoxin (fd), cyp153AP450 oxidase and ferredoxin reductase (fdR).The natural order of these genes and intergenic region is retained, but the GTG initiation codon derived from the cyp153A16 of Mycobacterium marinum is replaced by ATG initiation codon by overlapping PCR.Be cloned into by pcr amplification primer (amplimer) in pCL1920-derivative carrier (SC101 replicon, miramycin resistance marker), what make operon transcribes the control being subject to IPTG induction type Ptrc promoter.The plasmid pAS.017 of gained is (see table 11; vide infra) and pAS.018 be transformed in Escherichia coli MG1655, wherein fadE gene (encoding acyl-CoA dehydrogenasa) or fadD gene (encoding acyl-CoA synthase) deleted.These bacterial strains can not be degraded fatty acid, therefore can be realized the usability for increasing the fatty acid changing into product by the gene (such as fadA or fadB) deleting these genes or other Fatty acid degradation enzymes of encoding.But this is optional, and can be that external source feeds or implements for during the intermediate of product pathway at free fatty acid.(table 1 (as mentioned above) provides the extensive list thing of the enzymatic activity in described metabolic pathway, comprises and can be attenuated thus increase the multiple Fatty acid degradation enzyme of the usability of fatty acid in host strain.) bacterial strain of 4 kinds of gained is summarized in table 12 (vide infra).For the be converted bacterial strain analyze as described in of the dodecylic acid as described in embodiment 1 and 2 to sabinic acid.
MG1655 △ fadD (it does not express cyp153A operon) compared with control strain, in the GC-MS chromatogram of all 4 kinds of bacterial strains expressing cyp153A operon, detect the new peak (after BSTFA derivatization) (see Fig. 6, wherein bacterial strain sAS.320 and sAS.321 being only shown) at RT12.303 minute.Be shown in Fig. 7 A at the mass spectrum at the peak of RT12.303 minute.Fracture mode shows that this peak is 12-trimethylsiloxy dodecylic acid trimethyl silyl ester, and it is the derivatization form of sabinic acid.The characteristic ion fragment of 12-trimethylsiloxy fatty acid trimethyl silyl ester is shown in Fig. 8 A.At m/z=129, the ion of 147,204 and 217 (not shown, but exist) is the useful diagnostic flag of these compounds.Ion under m/z255 is used for determining to lose CH in carboxyl side 3after chain length (m/z=345) and (CH of hydroxyl side of this compound 3) 3siOH (see Fig. 8 A).The correct qualification at this peak is by comparing with the residence time and mass spectrum of the 12-trimethylsiloxy dodecylic acid trimethyl silyl ester true standard product shown in Fig. 7 B and be proved further.In addition, also have recorded the mass spectrum of this compound before derivatization, and be shown in Fig. 7 C.The characteristic ion fragment of the compound of underivatized is shown in Fig. 8 B.Ion under m/z=98 and 84 (display) is the useful diagnostic flag for ω-hydroxy fatty acid.Ion under m/z186 is that sabinic acid loses CH in hydroxyl side 2the fragments characteristic of O.By under m/z186 by CH 2it is 216 that O adds the molion that can measure described compound in fragmention.
Therefore, external source dodecylic acid is changed into sabinic acid in vivo by the Escherichia coli of expressing cyp153A operon (deriving from Marinobacteraquaeoli and Mycobacterium marinum) in host cell.Like this, can confirm that in fact described enzyme can produce ω-hydroxy fatty acid derivative.But the transformation efficiency of this enzyme is quite low.Therefore, estimate that the enzymatic activity of cyp153A operon itself is undesirable for production ω-hydroxy fatty acid derivative, and determine to need transformation further to design the enzyme with higher transformation efficiency.
Table 11: for the production of the expression plasmid of ω-hydroxylated derivative of fatty acid
Table 12: for derivative of fatty acid is changed into ω-hydroxylated derivative of fatty acid, express the recombinant escherichia coli strain of cyp153A
Bacterial strain Strain background Plasmid
sAS.314 MG1655 fadE fhuA pAS.017
sAS.315 MG1655 fadE fhuA pAS.018
sAS.320 MG1655 fadD pAS.017
sAS.321 MG1655 fadD pAS.018
sAS.335 MG1655 fadD pAS.022
sAS.336 MG1655 fadD pAS.023
Embodiment 4: dodecylic acid is changed into 12 (ω)-hydroxy-dodecanoic acids by the coli strain of expressing modified cyp153AP450 oxidase operon
The convert fatty acids that external source is added by the recombinant escherichia coli strain of expressing modified cyp153AP450 oxidase operon by the present embodiment display becomes ω-hydroxy fatty acid.Similarly, the object of this test is ability and the efficiency that this modified cyp153AP450 oxidase operon of research produces ω-hydroxy fatty acid derivative in vivo.
Described the mutant (wherein glycocoll 307 is replaced by alanine) (see HondaMalcaetal. (2012) Chem.Commun.48:5115) of the cyp153A obtained by M.aquaeoli before.Modify plasmid pAS.017 in such a way (see embodiment 3, as mentioned above): the codon defining the Gly307 of cyp153_Maqu becomes GCC from GGC, it defines Ala, in addition, IGR (TAAGGAGGAAAACAAA) (SEQIDNO:65) of synthesis is used to replace 2 of cyp156 operon natural intergenic regions (IGR).The plasmid called after pAS.022 of gained (see table 11, as mentioned above), and is converted in Escherichia coli MG1655 Δ fadD, thus obtains bacterial strain sAS.335 (see table 12, as mentioned above).Sabinic acid is changed into the bacterial strain (as mentioned above) as described in analyzing for the dodecylic acid as described in embodiment 1 and 2.As shown in Figure 9, compared with sAS.320, more dodecylic acid is changed into sabinic acid by sAS.335, and the amount of sabinic acid is defined as 37.4 ± 0.3mg/L.
Therefore, express the Escherichia coli display of modified cyp153A operon (deriving from Marinobacteraquaeoli) when feeding Exogenous Fatty Acid in vivo, external source dodecylic acid obtains certain improvement (compared with embodiment 3) to the conversion of sabinic acid.But the transformation efficiency of this enzyme is still lower.Therefore, estimate that the enzymatic activity of modified cyp153A operon (deriving from Marinobacteraquaeoli) itself is undesirable for production ω-hydroxy fatty acid derivative, and determine to need further transformation to design the enzyme with higher transformation efficiency.
Embodiment 5: derivative of fatty acid is changed into ω-hydroxylated derivative of fatty acid by the coli strain of expressing hybridization cyp153A-Red450RhF fused protein
Fatty acid, fatty acid methyl ester or fatty alcohol that external source is added by the recombinant escherichia coli strain of expressing hybrid proteins by the present embodiment display change into ω-hydroxy fatty acid, ω-hydroxy fatty acid methyl ester or α respectively, omega-diol, wherein cyp153AP450 oxidase and reductase domain fusion.The object of this test creates hybrid fusion protein matter, wherein cyp153AP450 oxidase with for significantly improveing the reductase domain fusion that ω-hydroxy fatty acid derivative is produced.
Self-centered Cytochrome P450 oxidase is the wherein enzyme that merges of reductase companion and Cytochrome P450 catalytic proteins.The P450RhF deriving from the bacterial classification NCIMB9784 of Rhod represents a class self-centered bacterial cytochrome P450 oxidase (Robertsetal. (2003) J.Biol.Chem.278:48914; Hunteretal. (2005) FEBSLett.579:2215), and become " PFOR that I class P450 merges " (DeMotandParret (2002) TrendsMicrobiol.10:502).
In this test, create the gene of coding hybrid fusion protein matter (obtain cyp153A (G307A) P450 catalytic proteins by Marinobacteraquaeoli and the P450RhF that obtained by the bacterial classification NCIMB9784 of Rhod form containing c-end FMN-and Fe/S reductase domain) in the following manner: to be increased cyp165A (G307A) _ Maqu gene by pAS.022, and by overlapping PCR and codon optimized synthesis P450RhF reductase domain fusion.The fusion (SEQIDNO:5) of gained is cloned in pCL1920-derivant (that is, SC101 replicon, miramycin resistance marker), it is transcribed be subject to the control of IPTG induction type Ptrc promoter.Described plasmid called after pAS.023 (see table 11, as mentioned above), and is converted in Escherichia coli MG1655 Δ fadD, thus obtains bacterial strain sAS.336 (see table 12, vide infra).
Then, sAS.336 bacterial strain is analyzed to sabinic acid and (iii) dodecanol to being converted of 1,12-dodecanediol to sabinic acid, (ii) dodecylic acid methyl ester for such as (i) dodecylic acid described in embodiment 1 and 2.Compared with control strain MG1655 △ fadD (as mentioned above), in GC/MS chromatogram, in sAS.336, identify the new peak (after BSTFA derivatization, see Figure 10 A to 10C) of all 3 kinds of compounds added.As described in Example 3, sabinic acid (as mentioned above) is identified.1,12-dodecanediol and sabinic acid methyl ester is accredited as respectively at the new peak of RT11.946 and RT11.668 minute (after BSTFA derivatization).
Figure 11 A shows the mass spectrum at the peak when RT11.948 minute.Ion fragmentation mode shows that this peak is two (trimethylsiloxy group) dodecane of 1,12-, and it is derived from 1,12-dodecanediol.Figure 13 A illustrates the ion fragmentation mode of two (trimethylsiloxy group) dodecane of 1,12-.At the fragments characteristic that the ion of m/z=147 is the compound that all glycol are correlated with, and at m/z=55, the ion of 69,83,97,111 and 125 is for losing HOSi (CH 3) 3the allylic scission fragments of characteristic after part.At the ion of m/z=241 for for determining α, the characteristic diagnostic flag of the chain length of omega-diol compound, as shown in FIG. 13A.The correct qualification at this peak is by comparing with the residence time and mass spectrum of 1,12-dodecanediol (using BSTFA+1%TMCS derivatization, Figure 11 B) true standard product and be proved further.Similarly, other α can be measured, the chain length of omega-diol.Such as, if peak has at the fragment of m/z269 with at m/z147, other specific ion of 149,111,97,83,69 and 55, then this peak is 1,14 pairs of (trimethylsiloxy group) tetradecanes.In order to identify undersaturated α, omega-diol, similar rule can be used.Due to undersaturated α, omega-diol 2 mass units fewer than their saturated counterpart, then the characteristic diagnosis fragment of 1,12-two (trimethylsiloxy group) dodecene is m/z=239.
Figure 12 A illustrates in order to the mass spectrum at peak when RT11.668 minute.According to the ion fragmentation mode of 12-trimethylsiloxy group dodecylic acid methyl ester, described peak is accredited as 12-trimethylsiloxy group dodecylic acid methyl ester.As in Figure 13 B prove, fragment into 2 kinds of main ions under described molecule, at m/z=287 (M-CH 3) and 255 (M-CH 3-HOCH 3).In addition, mass spectrum is presented at m/z=271 (M-OCH 3) characteristic ionic, show that this molecule has methyl ester moiety.Ion under m/z=103 shows that trimethylsiloxy group is in the position of end.In addition, the ion under m/z=287 and 255 is the characteristic ionic (see Figure 13 B) of the chain length determining this compound.Therefore, if peak has the fragment ion at m/z=315 and 283 and other ions at m/z55 and 103, then described peak can be accredited as 14-trimethylsiloxy group tetradecanoic acid methyl ester.Use this rule can identify the ω-hydroxy fatty acid methyl ester (see embodiment 6, vide infra) of the different chain length of being produced by bacterial strain stEP677.
Table 13 (vide infra) shows the amount of ω-hydroxylated derivative of fatty acid that bacterial strain sAS.336 transforms at 32 DEG C in 18h.As visible in table 13, compared with other checked enzymes, Exogenous fatty acid derivative is changed into ω-hydroxylated derivative of fatty acid (as mentioned above) by the hybridization cyp153A-RedRhF fused protein of being expressed by pAS.023 effectively.Therefore, select the enzyme of this transformation by reproducible feed, produce ω-hydroxy fatty acid derivative (see embodiment 6, vide infra) in vivo by the production host of transformation.
Table 13: the ω formed by sAS.336-hydroxylated derivative of fatty acid
* triplicate
Embodiment 6: by expressing the recombinant escherichia coli strain of CYP153A-Red450RhF hybrid fusion protein matter by glucose production ω-hydroxylated derivative of fatty acid
The recombinant escherichia coli strain of the present embodiment display by expressing chimeric hybrid proteins (wherein CYP153AP450 oxidase and reductase domain fusion), ω-hydroxy fatty acid, ω-hydroxy fatty acid methyl ester and α is produced, omega-diol by the renewable carbon hydrate feed of such as glucose and so on.
By the gene of pAS.023 amplification coding CYP153A (G307A)-RedRhF hybrid fusion gene, and be cloned into pACYC derivative carrier (p15a replicon, kalamycin resistance marks) in, what make fusion transcribes the control being subject to IPTG induction type Ptrc promoter.The plasmid called after pEP.125 of gained (see table 11, as mentioned above).
Use plasmid pAS.023 or pEP.125 transform respectively individually through transformation by 6 kinds of recombinant escherichia coli strains of the excessive production fatty acid of carbohydrate substrates (such as glucose) or derivative of fatty acid (see table 14, vide infra), thus create the bacterial strain (see table 15, vide infra) producing ω-hydroxylated derivative of fatty acid.In addition, the bacterial strain of excessive production fatty acid-or derivative of fatty acid also bacterial strain in contrast, thus easily identify the new compound in the bacterial strain producing ω-hydroxylated derivative of fatty acid.The bacterial strain of these excessive production fatty acid-or derivative of fatty acid describes in the present invention simply, but should not be interpreted as being limit.Create strains A LcV334 genome in such a way: delete fadE (acyl-CoA dehydrogenase) gene, and the variant of process LAN thioesterase tesA gene.Except carrying out except gene magnification in ALcV334, the genome of strain X L897 comprises following operation: the fatty acid biological operon for synthesizing (comprising the multiple genes described in table 1) of process LAN Phosphopantetheinyl transferase gene and synthesis; And integrate multiple operon, comprise carboxylate reductase (carB) variant; Thioesterase (tesA) variant; Alcohol dehydrogenase (AlrA); 3-ketone-acyl-acp synthase (fabB) variant; And transcription regulaton factor (fadR).Operate bacterial strain DAM1 genome in such a way: delete acyl-CoA dehydrogenase (fadE) gene, and process LAN Escherichia coli thioesterase (tesA) and acyl-CoA synthase (fadD) gene.
Operate bacterial strain stNH1293 in such a way, the genome of KASH286 and stNT29: delete acyl-CoA dehydrogenase (fadE) gene, and the fatty acid biological operon for synthesizing of process LAN transcription regulaton factor (fadR) and synthesis.In addition, bacterial strain stNH1293 comprises the plasmid of expressing plant thioesterase, acyl carrier protein (acp) gene and acetyl-CoA carboxylase (acc) gene composite.Bacterial strain KASH286 comprises the plasmid of expressing ester synthase variant.Bacterial strain stNT29 comprises the plasmid of expressing acyl ACP reductase (AAR) variant, alcohol dehydrogenase (AlrA), acyl carrier protein (acp) and acetyl-CoA carboxylase (acc) gene composite.
Table 14: the recombinant escherichia coli strain of excessive production fatty acid or derivative of fatty acid
Then, as described in embodiment 1 and 2, the ability of producing ω-hydroxylated derivative of fatty acid for the reproducible feed by such as glucose and so on analyzes the bacterial strain producing ω-hydroxylated derivative of fatty acid (as mentioned above) through transformation.ω-hydroxylated fatty acid, fatty acid methyl ester and fatty alcohol is identified as described in embodiment 3 and 5.
Table 15: for produced ω-hydroxylated derivative of fatty acid by reproducible carbohydrates feed, the recombinant escherichia coli strain of expressing cyp153A (G307A)-RedRhF fused protein
Table 16: recombinant escherichia coli strain is by the ω-hydroxylated derivative of fatty acid of glucose production
* triplicate
With control strain (see table 14, compare as mentioned above), qualification expresses cyp153A-RedRhF fused protein (see table 15, new peak (also see Figure 14 A-14C) in all bacterial strains of (corresponding to ω-hydroxy fatty acid, ω-hydroxy fatty acid methyl ester and α, omega-diol) as mentioned above).Table 16 shows these recombinant bacterial strains amount by the ω-hydroxy fatty acid derivative of glucose production in 20h.As table 16 is visible, the amount of the ω-hydroxy fatty acid derivative prepared by recombinant host cell is significant.Specifically, the production of discovery ω-hydroxy fatty acid derivative is very efficient.It should be noted that ω-hydroxy fatty acid that bacterial strain stEP675 produces almost finds (see Figure 15) only in supernatant, this shows that their product is released in supernatant by cell.Forming of ω-hydroxy fatty acid that Figure 16 display is produced by bacterial strain stEP675 and stEP682.The abundantest ω-hydroxy fatty acid that bacterial strain stEP682 produces is sabinic acid (79%).The abundantest ω-hydroxy fatty acid that 14-hydroxyl tetradecane acid (42%) is produced for stEP675.
In a word, bacterial strain stEP675 produces following ω-hydroxy fatty acid: 12-hydroxyl dodecenoic acid, sabinic acid, 14-hydroxyl tetradecenoic acid, 14-hydroxyl tetradecane acid, 16-hydroxy-16 carbon olefin(e) acid, juniperic acid and 18-hydroxyl 18 carbon diluted acid (see Figure 14 A).Bacterial strain stEP682 produces following ω-hydroxy fatty acid: 12-hydroxyl dodecenoic acid, sabinic acid, 14-hydroxyl tetradecenoic acid, 14-hydroxyl tetradecane acid, 16-hydroxy-16 carbon olefin(e) acid and juniperic acid.In addition, bacterial strain stEP677, except producing ω-hydroxy fatty acid, also produces following ω-hydroxy fatty acid methyl ester: sabinic acid methyl ester, 14-hydroxyl tetradecane acid methyl ester and juniperic acid methyl ester (see Figure 14 B).In addition, bacterial strain stEP684, except producing ω-hydroxy fatty acid, also produces following ω-hydroxy fatty acid methyl ester: 14-hydroxyl tetradecane acid methyl ester, 16-hydroxy-16 carbon olefin(e) acid methyl ester and juniperic acid methyl ester.Bacterial strain stEP676 produces following α, omega-diol: 1,12-dodecene glycol, 1,12-dodecanediol, 1,14-tetradecane diols, 1,16-cetene glycol and 1,16-hexadecane diol (Figure 14 C).Bacterial strain stEP685 produces following α, omega-diol: 1,14-tetradecane diols, 1,16-cetene glycol and 1,16-hexadecane diol.
It should be noted that the present embodiment display through transformation when the expression with CYP153A-RedRhF hybrid fusion protein matter is combined the coli strain of excessive production derivative of fatty acid effectively can produce ω-hydroxylated derivative of fatty acid by glucose (carbon source as unique).In addition, ω-hydroxylated fatty acid is secreted effectively in fermentation liquor (that is, producing cell or host cell by product secretion in fermentation liquor), and this is the desired characteristics of method of the present invention.
Embodiment 7: recombinant escherichia coli strain is by glucose production α, ω-diacid
The recombinant escherichia coli strain that the present embodiment proves to express chimeric hybrid proteins (wherein cyp153AP450 oxidase and reductase domain and alcohol oxidase and aldehyde dehydrogenase merge) produces α, ω-diacid by the reproducible carbohydrates feed of such as glucose and so on.
The gene of amplification coding alcohol oxidase alkJ (accession number CAB54054) (SEQIDNO:66) and aldehyde dehydrogenase alkH (accession number CAB51050) (SEQIDNO:68) is carried out by the genomic DNA of pseudomonas putida ATCC29347, and be cloned into pACYC derivative carrier (p15a replicon, kalamycin resistance marks) in, make 2 genes form operons, and this operon transcribe the control being subject to IPTG induction type Ptrc promoter.The plasmid called after pEP.126 of gained (see table 11, as mentioned above).By plasmid pEP126 and plasmid pAS.023, (see table 11, as mentioned above) cotransfection (see table 14, as mentioned above), thus obtains bacterial strain sEP690 to strains A lcV334.As described in embodiment 1 and 2, analyze bacterial strain for the ability by glucose production α, ω-diacid.Compared with control strain AlcV334, as shown in Figure 17, in sEP690, multiple new peak is identified.These peaks are accredited as α, the derivatization form of ω-diacid and α, ω-bis-(trimethyl silyl) fatty acid ester.α, the ion fragmentation mode of ω-bis-(trimethyl silyl) fatty acid ester and α, (it is α to ω-bis-(trimethylsiloxy group) fatty alcohol, the form of the derivatization of ω-diacid) very similar (see embodiment 3), difference is α, and ω-bis-(trimethyl silyl) fatty acid ester is not created on the ion of m/z=103.Therefore, this ion is for distinguishing α, ω-bis-(trimethylsiloxy group) fatty alcohol and α, ω-bis-(trimethyl silyl) fatty acid ester (and ω-1, ω-2 and ω-3 trimethylsiloxy group fatty alcohol, as mentioned below, vide infra) important information.
The main compound produced by bacterial strain stEP690 is 1,12-dodecanedioic acid, 1,14-carbon diluted acid, 1,14-tetracosandioic acid, 1,16-gaidic acid, 1,16-hexadecandioic acid (hexadecane diacid), 1,18-octadecene diacid (see Figure 17).The abundantest compound produced by this bacterial strain is 1, the 14-tetracosandioic acid when RT13.367 minute.Figure 18 A shows the mass spectrum of this compound after BSTFA derivatization.The molion of this compound is m/z=387 (M-CH 3), and its fracture mode is shown in Figure 19.By comparing with the mass spectrum and the residence time of true standard product 1, the 14-tetracosandioic acid after BSTFA derivatization, and prove 1, the qualification (Figure 18 B) of two (trimethyl silyl) ester of 14-tetracosandioic acid.
Table 16 shows the α that bacterial strain sEP690 is produced in 20h by glucose, the amount of ω-diacid, and it is ~ 550g/L.The display of this embodiment through transformation be used for excessive production derivative of fatty acid coli strain (when with the expression of CYP153A-RedRhF hybrid fusion protein matter in conjunction with time, and with the expression of alcohol oxidase (SEQIDNO:67) and aldehyde dehydrogenase (SEQIDNO:69) in conjunction with time) effectively produce α, ω-diacid by glucose (carbon source as unique).
Embodiment 8: produce the hydroxylated fatty acid of proximal end by the coli strain of expressing cyp102A1 (deriving from bacillus megaterium)
The object of this test is whether the host strain that research derives from the genetic modification of the F87A variant of the Cytochrome P450 cyp102A1 (P450-BM3) of bacillus megaterium by use expression produces ω-hydroxylated fatty acid or α, ω-diacid in vivo.Finding, a small amount of proximal end hydroxylated (ω-1, ω-2, ω-3, ω-4, ω-5) fatty acid can be produced by using the recombinant escherichia coli strain of expressing this cyp102A1 variant.But, ω-hydroxy fatty acid or α, ω-diacid can not be produced by using identical coli strain.
By bacillus megaterium genomic DNA (accession number AAA87602; SEQIDNO:61) overlapping PCR creates the gene that coding derives from the cyp102A1 genetic mutation (wherein the phenylalanine at position 87 place is replaced (F87A) by alanine) of bacillus megaterium (P450-BM3).By the DNA clone of amplification to pCL derivant (SC101 replicon, miramycin resistance marker) and pACYC derivant (p15a replicon, kalamycin resistance marks) in carrier, what make gene transcribes the control being subject to IPTG induction type Ptrc promoter.By plasmid pSN.012 and pSN.009 of gained (see table 17, vide infra) be converted into the strains A lcV334 of excessive production fatty acid (see table 14 and embodiment 16, as mentioned above), thus obtain bacterial strain sSN.012 and sSN.013 (see table 18, vide infra).Then, as described in embodiment 1 and 2, analyze these bacterial strains for these bacterial strains by the ability of glucose production ω-hydroxylated fatty acid.Compared with control strain AlcV334, in 2 kinds of bacterial strains, identify little new peak (in Figure 20 shown in sSN.012, after BSTFA derivatization).RT7.195 to 7.510 does not mate (see embodiment 3 with the mass spectrographic fracture mode at the new peak between 8.122 to 8.414 with ω-hydroxy fatty acid, see above), but, they and proximal end ω-1, ω-2, the expectation fracture mode coupling of the fatty acid hydroxyl group of ω-3, ω-4 and ω-5 position.Although the hydroxylated fatty acid of proximal end demonstrates the ion fragmentation mode similar to ω-hydroxy fatty acid (see Fig. 8 A, it is the sabinic acid of derivatization), these compounds show other fragment ion, this depends on m/z=117 (ω-1) after derivatization, 131 (ω-2), 145 (ω-3), the hydroxylated sites (see Figure 21 A-21E) of 159 (ω-4) or 173 (ω-5).About chain length, m/z=117 and 345 ion be combined as derivatization after 11 ω-1) specific ion of-hydroxy-dodecanoic acid.Similarly, at the specific ion being combined as ω-2, ω-3, ω-4 and ω-5 hydroxy-dodecanoic acid of m/z=131 and 331, m/z=145 and 317, m/z=159 and 303, m/z=173 and 289.Therefore, by the peak of RT7.195 to 7.510 be accredited as 7 (ω-5)-, 8 (ω-4)-, 9 (ω-3)-, 10 (ω-2)-and 11 (ω-1)-hydroxy-dodecanoic acids, and the peak of RT8.122 to 8.414 be accredited as 9 (ω-5)-, 10 (ω-4)-, 11 (ω-3)-, 12 (ω-2)-and 13 (ω-1)-hydroxy tetradecanoic acids.In addition, the existence for α, ω-diacid also carefully detects the GC/MS chromatogram of bacterial strain sSN.012 and sSN.013, but these bifunctional molecules do not detected.
To sum up, the present embodiment display through transformation be used for excessive production fatty acid coli strain when the F87A variant with the cyp102A1 obtained by bacillus megaterium expression in conjunction with time, do not produce ω-hydroxylated fatty acid, but produce the ω-1 of trace, ω-2, ω-3, ω-4 and ω-5 hydroxy fatty acid.Be reported in the cyp102A1 obtained by bacillus megaterium, F87A point mutation changes the substrate specificity of enzyme in vitro, makes the ω-position (see Oliveretal. (1997) Biochem.36:1567) of described enzyme almost special hydroxylation dodecylic acid or tetradecanoic acid.Report in addition, the P450-BM3 (cyp102A3) obtained by bacillus subtilis and P450-BM3 (F87A) (cyp153 (F87A)) produces 14-hydroxyl tetradecane acid and 1,14, tetracosandioic acid (for example, see WO2012/071439).But this not yet confirms, and seem to contradict with the present invention.In addition, about the data (such as Lentzetal. (2004) J.Biotechnol.108:41andLentzetal. (2006) ChemBioChem.7:345) that the Recent Report of the substrate specificity of the cyp102A3 (the close homologue of cyp102A1) of change bacillus subtilis supports the present invention to propose.Like this, phenylalanine residue of equal value in cyp102A3 is mutated into ω-1, ω-2, ω-3 and ω-4 position of the main hydroxylated fatty acid of valine (F88V), and in vitro, use dodecylic acid or hexadecanoic acid not observe the product of ω-hydroxylated as substrate.Therefore, the variant can concluding the mentioned above cyp102A1 obtained by bacillus megaterium and the cyp102A3 obtained by bacillus subtilis are not suitable for production ω-hydroxy fatty acid or α, ω-diacid (it needs ω-hydroxy fatty acid as intermediate).
Table 17: the expression plasmid built for the production of the hydroxylated derivative of fatty acid of proximal end
Plasmid Describe
pSN.009 PACYC-cyp102A1 (F87A) _ bacillus megaterium
pSN.012 PCL-cyp102A1 (F87A) _ bacillus megaterium
pHM105 PCL-cyp102A7_ bacillus licheniformis
pSL170.02 PCL-cyp102A7_ bacillus licheniformis-tesA*-alrA-fabB*-fadR
Table 18: the recombinant escherichia coli strain of expressing cyp102A protein produces the hydroxylated derivative of fatty acid of proximal end by reproducible carbohydrates feed
Embodiment 9: the coli strain of expressing cyp102A7 (deriving from bacillus licheniformis) produces ω-1, ω-2 and the hydroxylated fatty acid of ω-3-effectively by glucose
The object of the present embodiment is the hydroxylated fatty acid of large intestine bacterial classification production proximal end that Cytochrome P450-BM3-type oxidase cyp102A7 (deriving from bacillus licheniformis) is expressed in inspection.The coli strain of digital proof express cell cytochrome p 450-BM3-type oxidase cyp102A7 produces ω-1, ω-2 and ω-3-hydroxy fatty acid and fatty alcohol effectively.This is surprising and unexpected.
By bacillus licheniformis ATCC14580 genomic DNA (accession number AAU41718; SEQIDNO:63) the yrhJ gene (Dietrichetal. (2008) Appl.Microbiol.Biotechnol.79:931) of P450-BM3-type oxidase cyp102A7 that obtained by bacillus licheniformis of amplification coding.By this gene clone in pCL1920-derivant (SC101 replicon, miramycin resistance marker), make induction type Ptrc promoter control it and transcribe.In addition; by described gene clone in pCL1920-derivant; make induction type Ptrc promoter operon; it is by (successively) yrhJ; thioesterase variant (tesA); alcohol dehydrogenase (AlrA), 3-ketone-acyl-acp synthase variants (fabB) and transcription regulaton factor fadR form.Plasmid called after pHM105 and pSL170.02 respectively of gained, and be converted into (see table 14, described above) in coli strain LC972 and XL959.The present invention simply describes these bacterial strains (see also table 14).Transform the genome of bacterial strain LC972 in such a way: delete acyl-CoA dehydrogenase (fadE) gene.Phosphopantetheinyl transferase, the thioesterase variant (tesA) of 2 copies and fatty acid biological operon for synthesizing (the multiple genomic constitutions by described in the table 1) process LAN of synthesis.Strain X L959 is LC972, and it has the pACYC derivant plasmid of expressing carboxylate reductase carB variant.As described in embodiment 1 and 2, (see table 18, as mentioned above), difference is at the heated culture temperature of use 35 DEG C to analyze 4 kinds of new recombinant escherichia coli strain XL960-XL963 for the ability by the hydroxylated derivative of fatty acid of glucose production.Described bacterial strain all can not produce any ω-hydroxylated fatty acid or derivative of fatty acid.But as described in Example 8, all bacterial strains all produce the hydroxylated derivative of fatty acid of proximal end.Figure 22 A shows the chromatogram after the BFTSA derivatization obtained by strain X L961.As described in Example 8, described peak is accredited as following ω-1, ω-2 and ω-3-hydroxy fatty acid: 11-hydroxy-dodecanoic acid (RT=10.563), 10-hydroxy-dodecanoic acid (RT=10.512), 9-hydroxy-dodecanoic acid (RT=10.412), 13-hydroxyl tetradecane acid (RT=11.279), 12-hydroxyl tetradecane acid (RT=11.233), convolvulinolic acid (RT=11.133), 15-hydroxyl cetane acid (RT=11.679), 14-hydroxyl cetane acid (RT=11.623) and 13-hydroxyl cetane acid (RT=11.439).In addition, also have detected minimum peak, its most probable is equivalent to the hydroxylated fatty acid of undersaturated proximal end.
Figure 22 B shows the chromatogram after the BFTSA derivatization obtained by strain X L963.Described peak is accredited as following ω-1, ω-2 and ω-3-hydroxy fatty alcohols: at RT8.303, the peak of 8.480 and 8.572 minutes is respectively 7,8 and 9-hydroxyl decyl alcohol, at RT9.264, the peak of 9.653 and 9.905 is respectively 9,10 and 11-hydroxydodecanoic alcohol, at RT10.889, the peak of 11.044 and 11.124 is respectively 11, and 12 and 13-tetradecanol.For the identification of 9,10 and the detailed mass spectrum of 11-hydroxydodecanoic alcohol and ion fragmentation mode be shown in Figure 23 A, in 23B and 23C.Consider that the similar bacterial strain in embodiment 8 only produces the hydroxylated fatty acid of proximal end of trace, then wondrous and surprisingly all 4 kinds of bacterial strains all produce a large amount of these products (up to ~ 1.6g/L).Figure 24 A shows the amount of ω-1, ω-the 2 and ω-3-hydroxy fatty acid produced by strain X L960 and XL961, and Figure 24 B shows the amount of ω-1, ω-the 2 and ω-3-hydroxy fatty alcohols of being produced by strain X L962 and XL963.Therefore, the present embodiment display through transformation be used for when with the cyp102A7 that expression derives from bacillus licheniformis in conjunction with time excessive production derivative of fatty acid Escherichia coli, ω-1, ω-2 and ω-3-hydroxy fatty acid derivative is effectively produced by glucose (carbon source as unique).
Embodiment 10: recombinant escherichia coli strain is by glucose production α, ω-diester
Acyl-CoA synthase/ligase or transferase gene are cloned in pCL derivative carrier the downstream of the gene of cyp153A-RedRhF fused protein of encoding; make this 2 kinds of genes form operons, and this operon transcribe the control being subject to IPTG induction type Ptrc promoter.The example of suitable cyp153A and RedRhF fusion partner provides in table 2A and 2D.Suitable acyl-CoA synthase/ligase or the example of transferase provide in table 7.By the Plastid transformation of gained in the coli strain producing fatty acid methyl ester, such as KASH286 (see embodiment 6 and table 14, as mentioned above).As described in embodiment 1 and 2, analyze described bacterial strain for the ability by glucose production ω-hydroxylated derivative of fatty acid.Estimate that this bacterial strain can produce α, ω-diester.
Embodiment 11: recombinant escherichia coli strain is by glucose production omega-amino-derivative of fatty acid
By the gene clone of the gene of the gene of coding cyp153A-RedRhF fused protein, encoding alcohol oxidase or dehydrogenasa and coding aminopherase or transaminase in pCL derivative carrier, make this 3 kinds of genes form operons, and this operon transcribe the control being subject to IPTG induction type Ptrc promoter.The example of suitable cyp153A and RedRhF fusion partner provides in table 2A and 2D.Suitable alcohol oxidase or the example of dehydrogenasa provide in table 3A, and the example of suitable aminopherase or transaminase provides in table 4.By the Plastid transformation of gained in the coli strain producing fatty acid, such as stNH1293 (see embodiment 6 and table 14, as mentioned above).As described in embodiment 1 and 2, analyze described bacterial strain for the ability by glucose production ω-hydroxylated derivative of fatty acid.Estimate that this bacterial strain can produce omega-amino-fatty acid.Alternatively, by the Plastid transformation of gained in the coli strain producing fatty acid methyl ester, such as KASH286 (see embodiment 6 and table 14, as mentioned above).As described in embodiment 1 and 2, analyze described bacterial strain for the ability by glucose production ω-hydroxylated derivative of fatty acid.Estimate that this bacterial strain can produce omega-amino-fatty acid methyl ester.
Embodiment 12: produce ω-hydroxylated derivative of fatty acid by multiple feed
Because in nature, the biosynthesizing of fatty acid is general, so can implement embodiment provided by the present invention further in other organisms, wherein said organism uses the feed except carbohydrates natively.Such as these approach can be expressed in photosynthetic microbial organisms, thus allow by CO 2produce ω-hydroxylated derivative of fatty acid.Specifically, described approach can be expressed in the tenuigenin of cyanobacteria, wherein when described cyanobacteria grows under suitable condition (in such as bioreactor or open pond), they produce the ω-hydroxylated derivative of fatty acid that can be separated with culture.Alternatively, arbitrary technician of this area is clear that these approach can be expressed in the organism using carbon monoxide, such as, derive from those of fusobacterium.Such as when these through transformation microbial organisms in suitable condition (such as, in the reactor feeding the CO obtained by steel mill's waste gas or synthetic gas, wherein said synthetic gas is derivative by the improvement of organic material to be obtained, such as rock gas or living beings) under when growing, they can be produced can by the ω reclaimed in culture-hydroxylated derivative of fatty acid.
Embodiment 13: express the recombinant escherichia coli strain of multiple CYP153A-reductase hybrid fusion protein matter by glucose production ω-hydroxylated derivative of fatty acid
The recombinant escherichia coli strain of the chimeric hybrid proteins (wherein multiple CYP153AP450 oxidase domain and multiple reductase domain fusion) that the present embodiment Explicit Expression is other produces ω-hydroxy fatty acid by the reproducible carbohydrates of such as glucose and so on.
In this experiment, the gene of the reductase domain protein white matter that the gene of the CYP153AP450 catalytic proteins obtained by genomic DNA amplification coding 3 kinds of microorganisms and coding 4 kinds of microorganisms obtain, or be codon optimized DNA (seeing table 19) by these gene chemical synthesis.Reductase domain protein white matter comprises RedRhF-type and BM3-type protein.As described in Example 5, the gene of encoding chimera hybrid proteins assembled and be cloned into (as mentioned above) in pCL1920-derivative carrier.By the Plastid transformation of gained in strains A lcV334 (see table 14).Then, as described in embodiment 1 and 2, the ability of producing ω-hydroxylated derivative of fatty acid for the reproducible feed by such as glucose and so on analyzes these the 6 kinds of other bacterial strains producing ω-hydroxylated derivative of fatty acid through transformation.Following table 20 shows compared with StEP675, the amount of ω-hydroxylated fatty acid that these described bacterial strains are produced at 32 DEG C in 18h.StEP675 is the strains A lcV334 expressing chimeric protein, and wherein said chimeric protein is made up of (see embodiment 6) the CYP153A deriving from the water extra large bacillus of oil (M.aquaeolei) and the reductase domain of the bacterial classification NCIMB9784 deriving from Rhod.As visible in following table 20, most of bacterial strain of expressing CYP153A-reductase hybrid fusion protein matter produces ω-hydroxylated derivative of fatty acid effectively by glucose (carbon source as unique).To sum up, when chimeric CYP153A-reductase hybrid fusion protein matter is at expression in escherichia coli, effectively produce ω-hydroxylated derivative of fatty acid by glucose as unique carbon source, wherein said chimeric CYP153A-reductase hybrid fusion protein matter is made up of the protein of the different members of the different members and RedRhF-type or BM3-type reductase family that derive from CYP153A family.
Table 19: for produced ω-hydroxylated derivative of fatty acid by reproducible carbohydrates feed, the other recombinant escherichia coli strain of expressing hybridization cyp153A-reductase fused protein
Table 20: other recombinant escherichia coli strain is by the ω-hydroxylated derivative of fatty acid of glucose production
Following scheme and method are applicable to embodiment 14 to 19.
Scheme and method
Screening library
All schemes of the present invention all depend on the 96 orifice plates-deep-well plates-2mL system (GreinerBio-One for making culture grow, Monroe, NCorCorning, Amsterdam, TheNetherlands) and for by the flat board (Costar, Inc.) extracting fatty acid material in nutrient solution.Scheme provided below is the example of fermentation condition.Alternatives can be used to evaluate the production of fatty acid material.
The scheme that 32 DEG C of Plim cultivate:
Use 30 μ LLB cultures (being obtained by the LB culture grown in 96 orifice plates) to inoculate 290 μ LPlim nutrient culture media (following table 21), then, this nutrient culture media is shaken incubations about 16 hours at 32 DEG C.The seed using 40 μ L to spend the night inoculates 360 μ LPlim nutrient culture media.After 2 hours, IPTG (ultimate density is 1mM) Induced cultures (following table 21) is used 32 DEG C of growths.Then, if do not illustrated in addition, then culture is shaken incubations 20 hours at 32 DEG C, after this, extract these cultures according to the extraction scheme of standard hereinafter described.
35 DEG C of Nlim culture scheme:
Use 40 μ LLB cultures (being obtained by the LB culture grown in 96 orifice plates) to inoculate 360 μ LLB nutrient culture media (following table 21), then, this nutrient culture media is shaken incubations about 4 hours at 32 DEG C.40 μ LLB seeds are used to inoculate 360 μ LNlim nutrient culture media.After 2 hours, IPTG (ultimate density is 1mM) Induced cultures (following table 21) is used 32 DEG C of growths at 35 DEG C.Then, if do not illustrated in addition, then by culture at 35 DEG C of shakes=lower incubation 20 hours, after this, extract these cultures according to the extraction scheme of standard hereinafter described.
Table 21: the title of nutrient culture media and formula
The standard extraction scheme of fatty acid material:
To in each hole to be extracted, add 80 μ L1MHCl, then add 400 μ L butyl acetates (using 500mg/L pentadecanol as internal controls).Then, dull and stereotyped heat-sealing instrument (ALPS-300 well heater is used; Abgene, ThermoScientific, Rockford, IL) by 96 orifice plate heat-sealings, and use MIXMATE mixer (Eppendorf, Hamburg, Germany) to shake 15 minutes under 2000rpm.After shake, by flat board with 4500rpm in room temperature (AllegraX-15R, rotorSX4750A, BeckmanCoulter, Brea, CA) centrifugal 10 minutes, thus separate aqueous layer and organic layer.100 μ L organic layers are transferred in 96 orifice plates (polypropylene, Corning, Amsterdam, TheNetherlands), and use 100uLBSTFA to carry out derivatization.Subsequently flat board is sealed, and at being stored in-20 DEG C, until use w-OHFFA method to be evaluated by GC-FID in such a way: by 1 μ L Sample Injection to Agilent7890AGCUltra device (Agilent, SantaClara, CA) analytical column (DB-1,10m × 180 μm × 0.2 μM of thickness, derives from JW121-101A) on, wherein said Agilent7890AGCUltra device has the flame ionization detector (FID) of 1-20 shunting.Instrument described in foundation is used for detecting and quantitative C 10to C 18fatty acid and ω-hydroxy fatty acid.The scheme of above-detailed represents the condition of standard, and it can be revised as required with Optimization analyses result.
Set up fallibility library
The known standard technique of those skilled in the art is used to prepare fallibility library.In one embodiment, under the condition being conducive to the nucleotide introducing mispairing, when carrying out diversity establishment by DNA profiling by pcr amplification in DNA embolus, the restriction endonuclease in carrier is used to prepare the skeleton of carrier.In one approach, use INFUSIONCloningSystem (ClontechLaboratories, Inc., MountainView, CA), carry out carrier framework according to the scheme of manufacturer and there is the clone of multifarious DNA embolus.
Set up saturated library
The known standard technique of those skilled in the art is used to prepare saturated library.In one embodiment, when using degraded primer to carry out diversity establishment in DNA embolus, the restriction endonuclease in carrier is used to prepare carrier framework.In one approach, use INFUSIONCloningSystem (ClontechLaboratories, Inc., MountainView, CA), carry out carrier framework according to the scheme of manufacturer and there is the clone of multifarious DNA embolus.
Set up combinatorial libraries
To favourable mutation combination be identified as, thus provide the production of ω-OH derivative of fatty acid material to obtain the CYP153-reductase hybrid fusion polypeptide variants (such as CYP153A-RedRhF hybrid proteins variant) of improvement further.The known standard technique of those skilled in the art is used to prepare combinatorial libraries.In one embodiment, when using the primer introducing required sudden change to carry out diversity establishment in DNA embolus, the restriction endonuclease in carrier is used to prepare carrier framework.As described above, in one approach, INFUSIONCloningSystem (ClontechLaboratories is used, Inc., MountainView, CA), carry out carrier framework according to the scheme of manufacturer and there is the clone of multifarious DNA embolus.Transfer PCR (tPCR) scheme (Erijmanetal. (2011) J.StructuralBio.175:171-177) can be used to generate combinatorial libraries.
Library screening
Once generate library diversity in fallibility library, saturated library or combinatorial libraries after, just use a kind of method mentioned above to screen these libraries.Identify the situation that meets (hit) of 2 types: the amount (ω OHFFA tires) of (1) ω-hydroxy fatty acid increases; And/or (2) fatty acid increases to the conversion of ω-hydroxy fatty acid.Use the standard technique that those skilled in the art commonly use, identified the sudden change of the various hybridization cyp153A-RedRhF protein variants met in situation by order-checking.Following table 23,24 and 25 lists in saturated library and is identified as favourable sudden change (meeting situation).
Embodiment 14: for bacterial strain and the plasmid construction of library screening
Present embodiment describes for saturated or combinatorial mutagenesis library screening and the bacterial strain built and plasmid.
Create the gene for hybrid fusion protein matter of encoding (by deriving from CYP153A (G307A) the P450 catalytic proteins of Marinobacteraquaeoli and deriving from the preparing containing the reductase domain of c-end FMN-and Fe/S of P450RhF of bacterial classification NCIMB9784 of Rhod) in such a way: by genomic DNA amplification cyp165A (G307A) _ Maqu gene, and by overlapping PCR and codon optimized synthesis P450RhF reductase domain fusion.Described fusion (SEQIDNO:5) is cloned in pACYC-derivant (that is, p15A replicon, kalamycin resistance mark), it is transcribed be subject to the control of IPTG induction type Ptrc promoter.Described plasmid called after pEP125 (see table 22, vide infra).In addition; by the gene of pEP125 amplification coding CYP153A (G307A)-Red450RhF hybrid fusion protein matter; and be cloned into pCL1920-derivative carrier (SC101 replicon; miramycin resistance marker) in; it is transcribed be subject to the control of IPTG induction type Ptrc promoter; and which form operon, this operon has the gene of coded plant thioesterase (fatB1), 3-ketone-acyl-acp synthase variants (fabB) and transcription regulaton factor (fadR).Described plasmid called after pLC81 (see table 22, vide infra).
Create other plasmid in such a way: be codon optimized DNA by the gene chemical synthesis of coded plant thioesterase (fatB1) (deriving from California bay); and be cloned into pCL1920-derivative carrier (SC101 replicon; miramycin resistance marker) in; it is transcribed be subject to the control of IPTG induction type Ptrc promoter; and which form operon, this operon has the gene of encoding acetyl base-CoA carboxylase (accDACB), biotin ligase (birA) and acyl carrier protein.Described plasmid called after pNH305 (see table 22, vide infra).Plant thioesterase (fatA3) (the deriving from arabidopsis) of the synthesis optimized by the son that accesses to your password replaces the fatB1 in pNH305, creates plasmid pAS033 (see table 22, vide infra).Plant thioesterase (fatA3) (the deriving from arabidopsis) of the synthesis optimized by the son that accesses to your password replaces fatB1 in pLC81, creates plasmid pEP146 (see table 22, vide infra).In addition, pEP146 also carries the sudden change in plasmid-encoded repA protein.
Basic bacterial strain for Plastid transformation is GLP077 and BZ128.In brief, operate the genome of basic bacterial strain GLPH077 in such a way: delete acyl-CoA dehydrogenase (fadE) gene, and the fatty acid biological operon for synthesizing of transcription regulaton factor (fadR) and synthesis is over-expressed.In brief; operate the genome of basic bacterial strain BZ128 in such a way: delete fadE (acyl-CoA dehydrogenase) gene, and the fatty acid biological operon for synthesizing of synthesis, beta-hydroxy fatty acyl group-ACP dehydratase (fabZ) and thioesterase variant (tesA) are over-expressed.In addition, described bacterial strain is in advance through swivel base (transposon) and MNNG (NTG) mutagenesis and screening.
Table 22: for the plasmid of library screening
Plasmid Describe
pAS033 pCL-fatA3_Atal-accDCBAbirA_Cglu-acp_Ecol
pEP125 pACYC-cyp153A(G307A)_Maqu-RedRhF_Rhod
pNH305 pCL-fatB1_Ucal-accDCBAbirA_Cglu-acp_Ecol
pLC81 pCL-cyp153A(G307A)_Maqu-RedRhF_Rhod-fatB1_Ucal-fadB_Ecol-fadR_Ecol
pEP146 pCL*-cyp153A(G307A)_Maqu-RedRhF_Rhod-fatA3-Atal-fadB_Ecol-fadR_Ecol
Inspection hybridization cyp153A (G307A)-Red450RhF fused protein, to check whether the expression in host cell can produce ω-OH derivative of fatty acid.The microbial organisms of expressing SEQIDNO:5 can by the ω-OH derivative of fatty acid of glucose production more than 1g/L.Therefore, select the enzyme of this transformation for further developing research.
The saturated library of the P50 catalyst structure domain of embodiment 15:cyp153A (G307A)-Red450RhF fused protein
Set up the complete saturated library of the P450 catalyst structure domain of CYP153A-Red450RhF fused protein, and screen for variant, wherein said variant display is more than the improvement of CYP153A (G307A)-Red450RhF (that is, template peptide).The favourable sudden change (see HondaMalcaetal. (2012) Chem.Commun.48:5115) that G307A (glycine residue replaces the alanine at position 307 place) is the ω-hydroxylase activity of improvement CYP153A.The choice criteria meeting situation is that the amount of (1) ω-hydroxy fatty acid (ω OHFFA tires) increases; And/or (2) fatty acid to ω-hydroxy fatty acid conversion increase.
The known standard technique of those skilled in the art is used to prepare saturated library.Plasmid pEP125 and pLC81 is used (see table 22, as mentioned above) to prepare complete saturated library.Screen 3 kinds of saturated libraries: for the first library, pEP125 and pNH305 is converted in bacterial strain GLPH077 together; For the second library, pLC81 is converted in BZ128; And for the 3rd library, pEP125 and pAS.033 is converted in GLPH077 bacterial strain together.Obtain in the formation specifically for ω-hydroxy-dodecanoic acid screening the first and second libraries in the variant improved, and obtain screening the 3rd library in the variant improved in the formation specifically for ω-hydroxy-16 carbon olefin(e) acid.Use the scheme of a kind of standard mentioned above to screen described library.The variant of improvement is shown in following table 23 to 25 and (vide infra).Specifically, repeatedly identify the variant at position 141 place, and find that it is the enzyme of the remarkable improvement forming ω-hydroxy-dodecanoic acid and ω-hydroxy-16 carbon olefin(e) acid.
Table 23: the general introduction of the improved variants obtained by the first saturated library, site of the catalyst structure domain of CYP153A (G307A)-Red450RhF
FIOC: the multiple exceeding the improvement of contrast; Contrast is runic.
Table 24: the general introduction of the improved variants obtained by the second saturated library, site of the catalyst structure domain of CYP153A (G307A)-Red450RhF
FIOC: the multiple exceeding the improvement of contrast; Contrast is runic.
Table 25: the general introduction of the improved variants obtained by the 3rd saturated library, site of the catalyst structure domain of CYP153A (G307A)-Red450RhF
FIOC: the multiple exceeding the improvement of contrast; Contrast is runic.
The saturated library of moiety site of the reductase domain of embodiment 16:CYP153A (G307A)-Red450RhF fused protein
Set up the fractional saturation library (every 10th amino acid is suddenlyd change) of the reductase domain of hybridization CYP153A-Red450RhF fused protein, and screen for the variant that such as goes down, wherein said variant display is more than CYP153A (V141I, A231T, G307A) (it is the improvement of the variant identified in the saturation mutagenesis library, site of catalysis P450CYP153A domain to-Red450RhF (SEQIDNO:32).Increase for meeting the amount (ω OHFFA tires) that the choice criteria of situation is (1) ω-hydroxy-dodecanoic acid; And/or (2) dodecylic acid increases to the conversion of ω-hydroxy-dodecanoic acid.The known standard technique of those skilled in the art is used to prepare saturated library.For described library, the pLC81 that will take CYP153A (V141I, A231T, G307A)-Red450RhF in is converted in BZ128.Use a kind of standard scheme mentioned above to screen library.The variant of improvement is shown in following table 26.Specifically, modification A 796V (SEQID:42) and P666A is the enzyme of restriction improvement.
Table 26: the general introduction of the improved variants obtained by the fractional saturation library of the reductase domain of CYP153A (V141IA231TG307A)-Red450RhF
FIOC: the multiple exceeding the improvement of contrast; Contrast is runic.
The combinatorial libraries of the reductase domain of embodiment 17:CYP153A (G307A)-Red450RhF fused protein
The favourable sudden change (embodiment 17) identified in the fractional saturation library of reductase domain is the basis of the combinatorial libraries improveing CYP153A (G307A)-Red450RhF fused protein further.The amount (ω OHFFA tires) that choice criteria is (1) ω-hydroxy-dodecanoic acid increases; And/or (2) dodecylic acid increases to the conversion of ω-hydroxy-dodecanoic acid.
In the pLC81 taking CYP153A (V141I, A231T, G307A)-Red450RhF (SEQID:32) in, build combinatorial libraries, and be converted in BZ128.The known standard technique of those skilled in the art is used to prepare combinatorial libraries.Use a kind of standard scheme mentioned above to screen library.The variant of improvement is shown in following table 27.
Table 27: the general introduction of the improved variants obtained by the combinatorial libraries of the reductase domain of CYP153A (V141I, A231T, G307A)-Red450RhF
FIOC: the multiple exceeding the improvement of contrast; Contrast is runic.
The catalysis of embodiment 18:CYP153A (G307A)-Red450RhF fused protein and the combinatorial libraries of reductase domain
The favourable sudden change (embodiment 16 and 17) identified in saturated library is the basis of the combinatorial libraries improveing CYP153A (G307A)-Red450RhF fused protein further.The amount (ω OHFFA tires) that choice criteria is (1) ω-hydroxy-dodecanoic acid increases; And/or (2) dodecylic acid increases to the conversion of ω-hydroxy-dodecanoic acid.In pLC81, build combinatorial libraries, and be converted in BZ128.The known standard technique of those skilled in the art is used to prepare combinatorial libraries.Use a kind of standard scheme mentioned above to screen library.2 kinds of best improved variants are shown in Table 28.
Table 28: the best improved variants obtained by the combinatorial libraries of CYP153A (G307A)-Red450RhF
* tire (mg/L) after 48h
The site saturation mutagenesis of the position 141 and 309 of embodiment 19:CYP153A (G307A, A796V)-Red450RhF
Notice the variable effect substrate specificity at position 141 place.Therefore, in CYP153A (G307A, A796V)-Red450RhF at this enforcement site, 2 positions saturation mutagenesis.The choice criteria meeting situation is the amount increase of (1) ω-hydroxy-16 carbon olefin(e) acid; And/or (2) gaidic acid increases to the conversion of ω-hydroxy-16 carbon olefin(e) acid.
For described library, the pEP146 taking CYP153A (G307AA796V)-Red450RhF (SEQID:38) in is converted in BZ128.The known standard technique of those skilled in the art is used to prepare saturated library, site.Use a kind of standard method mentioned above to screen library.Specifically, the variant (SEQID:46) with V141T demonstrates the highest ω-hydroxy-16 carbon olefin(e) acid and to tire and by the highest conversion (Figure 27) of gaidic acid.
Embodiment 20: the recombinant escherichia coli strain of expressing the hybridization CYP153A-Red450RhF fused protein of improvement produces ω-hydroxylated fatty acid by glucose high-titer
The recombinant escherichia coli strain of the hybridization CYP153A-Red450RhF fused protein of the present embodiment Explicit Expression improvement produces ω-hydroxy fatty acid by the renewable carbon hydrate feed high productivity of such as glucose and so on.
The gene (SEQIDNo:46) of encode variant being hybridized CYP153A-Red450RhF fused protein is cloned into pCL1920-derivative carrier (the SC101 replicon of modification; miramycin resistance marker) in; it is transcribed be subject to the control of IPTG induction type Ptrc promoter, and which form the operon with plant thioesterase (fatA3), 3-ketone-acyl-acp synthase variants (fabB) and transcription regulaton factor (fadR).By described Plastid transformation in bacterial strain L439, thus obtain bacterial strain stEP.798.In brief, the genome of basic bacterial strain L439 comprises following operation: delete fadE (acyl-CoA dehydrogenase) gene, and the fatty acid biological operon for synthesizing of synthesis and thioesterase variant (tesA) are over-expressed.In addition, described bacterial strain is in advance through swivel base and MNNG (NTG) mutagenesis and screening.
Described bacterial strain is run in such a way: cultivated in the LB shaking flask comprising miramycin (115mg/L) at 32 DEG C by the cell bank bottle of described bacterial strain, until the OD reading >1 of culture in bio-reactor.This culture of 5%v/v to be transferred in FA seed culture medium (2g/L ammonium chloride, 0.5g/L sodium chloride, 0.3g/L potassium dihydrogen phosphate, 1mM magnesium sulfate, 0.1mM lime chloride, 20g/L glucose, 1mL/L trace element solution, 10mg/L Citric Acid Mono iron, 100mMbis-tris damping fluid and 115mg miramycin) and 32 DEG C of incubated overnight.Then, use this inoculum inoculate for the production of preparative bio-reactor.Initial bioreactor culture base for this technique comprises: 0.5g/L ammonium chloride, 1g/L sodium chloride, 4g/L potassium dihydrogen phosphate, 2.2g/L epsom salt, 140mg/L calcium chloride dihydrate, 10mL/L trace element solution, 80mg/L Citric Acid Mono iron, 0.6mL/L trace vitamin solution and 5g/L primverose starch.After the sterilizing of bio-reactor, admixture comprises: 0.2mM amino-laevulic acid, 30g/L glucose and 115mg/L miramycin.
Before inoculation, the parameter of stabilate reactor and control loop is opened-dissolved oxygen DO setting value: 30%; Desired temperature: 29 DEG C; Aeration set value: 0.5vvm; PH setting value: 6.9.When the density of culture is higher than OD30, uses 5%v/v inoculum inoculation bio-reactor, and use 1mMIPTG to induce.DO trigger (when the glucose in nutrient culture media is depleted, described DO trigger makes instruction to controller) is used complex glucose feed solution (586g/L glucose, 2.2g/L epsom salt, 0.4g/L potassium dihydrogen phosphate, 80mg/L Citric Acid Mono iron and 10mL/L trace element solution) to be fed in culture with maximum rate 10g/L glucose (according to nominal culture volume).To bio-reactor sampling in the whole process run, and gather in the crops after 72 hours in cultivation.
Figure 25 shows the amount of the ω-hydroxylated fatty acid produced by bacterial strain stEP.798 in 30.5 DEG C of whole processes at 72h, and it reaches the high 16.0g/L that tires after 72h.The ω produced-hydroxylated fatty acid is made up of 63.1% ω-hydroxy-16 carbon olefin(e) acid (C16:1), 26.4% ω-hydroxyl cetane acid (C16:0), 7.6% ω-hydroxyl tetradecane acid (C14:0), 1.9% ω-hydroxyl tetradecenoic acid (C14:1) and a small amount of ω-hydroxyl dodecenoic acid (C12:0) and ω-hydroxyl dodecenoic acid (C12:1) (C12 is lower than 1%).In addition, stEP.798 produces 3.0g/L fatty acid when 72h.To sum up, the expression of hybridization CYP153A-Red450RhF fused protein in the coli strain producing fatty acid of improvement can produce ω-hydroxylated fatty acid by reproducible carbohydrates feed high-titer.
Embodiment 21: the recombinant escherichia coli strain of expressing the hybridization CYP153A-Red450RhF fused protein of improvement produces α by glucose high-titer, ω-diacid
The hybridization CYP153A-Red450RhF fused protein of the present embodiment Explicit Expression improvement and the recombinant escherichia coli strain of allos alcohol oxidase (alkJ) and aldehyde dehydrogenase (alkH) produce α, ω-diacid by the renewable carbon hydrate feed high productivity of such as glucose and so on.
The gene (SEQIDNo:42) of encode variant being hybridized CYP153A-Red450RhF fused protein is cloned into pCL1920-derivative carrier (the SC101 replicon of modification; miramycin resistance marker) in; it is transcribed be subject to the control of IPTG induction type Ptrc promoter, and which form the operon with plant thioesterase (fatB1), alcohol oxidase (alkJ), aldehyde dehydrogenase (alkH), 3-ketone-acyl-acp synthase variants (fabB) and transcription regulaton factor (fadR).By described Plastid transformation in bacterial strain L1012, thus obtain bacterial strain L1017.In brief; the genome of basis bacterial strain L1012 comprises following operation: delete fadE (acyl-CoA dehydrogenase) and adhE (alcohol dehydrogenase) gene, and the fatty acid biological operon for synthesizing of synthesis, beta-hydroxy fatty acyl group-ACP dehydratase (fabZ) and thioesterase variant (tesA) are over-expressed.In addition, described bacterial strain is in advance through swivel base and MNNG (NTG) mutagenesis and screening.
Described bacterial strain is run in such a way: cultivated in the LB shaking flask comprising miramycin (115mg/L) at 32 DEG C by the cell bank bottle of described bacterial strain, until the OD reading >1 of culture in bio-reactor.This culture of 2%v/v to be transferred in FA seed culture medium (2g/L ammonium chloride, 0.5g/L sodium chloride, 0.3g/L potassium dihydrogen phosphate, 1mM magnesium sulfate, 0.1mM lime chloride, 20g/L glucose, 1mL/L trace element solution, 10mg/L Citric Acid Mono iron, 100mMbis-tris damping fluid and 115mg miramycin) and 32 DEG C of incubated overnight.Then, use this inoculum inoculate for the production of preparative bio-reactor.Initial bioreactor culture base for this technique comprises: 0.5g/L ammonium chloride, 1g/L sodium chloride, 4g/L potassium dihydrogen phosphate, 2.2g/L epsom salt, 140mg/L calcium chloride dihydrate, 10mL/L trace element solution, 80mg/L Citric Acid Mono iron, 0.6mL/L trace vitamin solution and 5g/L primverose starch.After the sterilizing of bio-reactor, admixture comprises: 0.2mM amino-laevulic acid, 30g/L glucose and 115mg/L miramycin.
Before inoculation, the parameter of stabilate reactor and control loop is opened-dissolved oxygen DO setting value: 30%; Desired temperature: 31 DEG C; Aeration set value: 0.5vvm; PH setting value: 6.9.When the density of culture is higher than OD30, uses 5%v/v inoculum inoculation bio-reactor, and use 1mMIPTG to induce.PH trigger (when the glucose in nutrient culture media is depleted, described pH trigger makes instruction to controller) is used complex glucose feed solution (586g/L glucose, 2.2g/L epsom salt, 0.4g/L potassium dihydrogen phosphate, 80mg/L Citric Acid Mono iron and 10mL/L trace element solution) to be fed in culture with 10g/L group's material (according to nominal culture volume).To bio-reactor sampling in the whole process run, and gather in the crops after 48 hours of incubation.
Figure 26 shows in 30.5 DEG C of whole processes at 48h, the amount of the α produced by bacterial strain L1017, ω-diacid.Described bacterial strain produces 21.2g/L α, ω-diacid when 31h, and after 48h, reach the high 23.9g/L that tires.The α produced, ω-diacid is by 85.9% α, ω-dodecylic acid (C12:0), 4.7% α, ω-dodecenoic acid (C12:1), 5.7% α, ω-tetradecanoic acid (C14:0), 2.9% α, ω-tetradecenoic acid (C14:1) and a small amount of α, ω-gaidic acid (C16:1) (C16 is lower than 1%) composition.In addition, stEP.798 produces 9.3g/L fatty acid when 48h.ω-the hydroxy fatty acid of trace only detected.To sum up, the expression of hybridization CYP153A-Red450RhF fused protein in the coli strain of excessive production fatty acid of improvement and make it possible to produce α, ω-diacid by reproducible carbohydrates feed high-titer with the coexpression of alcohol oxidase (alkJ) and aldehyde dehydrogenase (alkH).
To it will be apparent to those skilled in the art that under the condition not departing from spirit and scope disclosed by the invention can in above-mentioned and embodiment carry out multiple amendment and change.This type of amendment and change are within scope disclosed by the invention.

Claims (52)

1. under the carbon source obtained by reproducible feed exists, when growing in fermentation liquor, for producing the recombinant microorganism organism of ω-hydroxy fatty acid derivative in vivo, described microbial organisms comprises the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises:
The thioesterase of (a) EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And
Modified ω-the hydroxylase of (b) EC1.14.15.3.
2. recombinant microorganism organism according to claim 1, wherein said modified ω-hydroxylase has the enzymatic activity of modified cytochrome P 450 monooxygenases, and the ω position of catalytic hydrocarbon chain effectively in vivo.
3. recombinant microorganism organism according to claim 2, wherein said modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.
4. recombinant microorganism organism according to claim 3, wherein said CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.
5. recombinant microorganism organism according to claim 1, wherein said modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.
6. recombinant microorganism organism according to claim 5, wherein said ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and have the one or more sudden changes be selected from V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and A796V.
7. recombinant microorganism organism according to claim 1, wherein said recombinant microorganism organism produces the ω-hydroxy fatty acid derivative be selected from ω-hydroxy fatty acid and ω-hydroxy fatty acid methyl ester.
8. recombinant microorganism organism according to claim 1, wherein said recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.1.1/2, or the alcohol oxidase of EC1.1.3.13 or EC1.1.3.20.
9. recombinant microorganism organism according to claim 8, wherein said recombinant microorganism organism produces the ω-hydroxy fatty acid derivative be selected from ω-oxo fatty acid and ω-oxo fatty acid methyl ester.
10. recombinant microorganism organism according to claim 8, wherein said recombinant microorganism organism is encoded further through transformation the nucleotide sequence of following polypeptide, and described polypeptide comprises the aldehyde dehydrogenase of EC1.2.1.3/4/5 or the aldehyde oxidase of EC1.2.3.1.
11. recombinant microorganism organisms according to claim 10, wherein said recombinant microorganism organism produces ω-hydroxy fatty acid derivative, and this ω-hydroxy fatty acid derivative is α, ω-diacid or ω-carboxylic fatty acids methyl ester.
12. recombinant microorganism organisms according to claim 10; wherein said recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises acyl group-CoA ligase or the EC2.8.3.6 acyl group-CoA transferase of EC6.2.1.3.
13. recombinant microorganism organisms according to claim 12, wherein said recombinant microorganism organism produces ω-hydroxy fatty acid derivative, and this ω-hydroxy fatty acid derivative is α, ω-diester.
14. recombinant microorganism organisms according to claim 8, wherein said recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the aminopherase of EC2.6.1 or the amine dehydrogenase of EC1.4.9, EC1.4.98 or EC1.4.99.
15. recombinant microorganism organisms according to claim 14, wherein said recombinant microorganism organism produces ω-hydroxy fatty acid derivative, and this ω-hydroxy fatty acid derivative is selected from omega-amino-fatty acid and omega-amino-fatty acid methyl ester.
16. recombinant microorganism organisms according to claim 1, wherein said recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.-.-and the carboxylate reductase of 1.2.99.
17. recombinant microorganism organisms according to claim 16, wherein said ω-hydroxy fatty acid derivative is α, omega-diol.
18. cell cultures comprising the microbial organisms according to any one of claim 1-17.
19. cell cultures according to claim 18, wherein said cell culture produces ω-hydroxy fatty acid derivative, and this ω-hydroxy fatty acid derivative is selected from ω-hydroxyl free fatty acid; ω-hydroxy fatty acid methyl ester; ω-oxo fatty acid; ω-oxo fatty acid methyl ester; α, ω-diacid; α, omega-diol; Omega-amino-fatty acid; And omega-amino-fatty acid methyl ester.
The method of 20. production ω-hydroxy fatty acid derivatives, it comprises:
A () provides recombinant microorganism organism in fermentation liquor, described microbial organisms comprises the approach of at least 2 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the thioesterase of EC3.1.2.-, 3.1.1.5 or 3.1.2.14; Or the ester synthase of EC2.3.1.75 or EC2.3.1.20; And the modified ω-hydroxylase of EC1.14.15.3;
B the reproducible feed comprising carbon source adds in described fermentation liquor by (); And
C () is by described separation of fermentative broth ω-hydroxy fatty acid derivative.
21. methods according to claim 20, wherein said modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.
22. methods according to claim 21, wherein said CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.
23. methods according to claim 20, wherein said modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.
24. methods according to claim 23, wherein said ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and have the one or more sudden changes be selected from V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and A796V.
25. methods according to claim 20, wherein said ω-hydroxy fatty acid derivative is ω-hydroxyl free fatty acid or ω-hydroxy fatty acid methyl ester.
26. methods according to claim 20, wherein said recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the alcohol dehydrogenase of EC1.1.1.1/2 or the alcohol oxidase of EC1.1.3.13 or EC1.1.3.20.
27. methods according to claim 26, wherein said ω-hydroxy fatty acid derivative is ω-oxo fatty acid or ω-oxo fatty acid methyl ester.
28. methods according to claim 26, wherein said recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the aldehyde dehydrogenase of EC1.2.1.3/4/5 or the aldehyde oxidase of EC1.2.3.1.
29. methods according to claim 28, wherein said ω-hydroxy fatty acid derivative is α, ω-diacid or α, omega-3-fatty acids dimethyl esters.
30. methods according to claim 26, wherein said recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the aminopherase of EC2.6.1 or the amine dehydrogenase of EC1.4.9, EC1.4.98 or EC1.4.99.
31. methods according to claim 30, wherein said ω-hydroxy fatty acid derivative is omega-amino-fatty acid or omega-amino-fatty acid methyl ester.
32. methods according to claim 20, wherein said recombinant microorganism organism expresses the nucleotide sequence of following polypeptide of encoding further through transformation, described polypeptide comprises the carboxylate reductase of EC1.2.99.6 or the alcohol dehydrogenase of EC1.1.-.-.
33. methods according to claim 32, wherein said ω-hydroxy fatty acid derivative is α, omega-diol.
34. methods according to any one of claim 1-33, wherein said reproducible feed is carbon back.
35. methods according to claim 34, the reproducible feed of wherein said carbon back is selected from corn, sugarcane, Chinese sorghum, beet, switchgrass, fresh stover, straw, timber, paper pulp, sewage, rubbish, cellulose municipal waste, waste gas, synthetic gas, biomass hydrolysate and carbon dioxide.
36. methods according to claim 1, wherein said carbon source is selected from glucose, fructose, mannose, galactose, wood sugar, arabinose, FOS, galactooligosaccharide, starch, cellulose, colloid, xylan, sucrose, maltose, cellobiose, turanose, hemicellulose, methylcellulose, sodium carboxymethyl cellulose, succinate, lactate, acetic acid esters, ethanol, methyl alcohol, glycerine, refuse fatty acid and their potpourri.
37. methods according to claim 1, wherein said carbon source is glucose, glycerine or sucrose.
38. polymer compositions produced by method according to claim 20, wherein said polymer composition is selected from polyurethane, polyester polyol, vibrin, alkyl coating resin, glass fiber resin, gel coat resin and thermoplastic polyester.
39. under the carbon source obtained by reproducible feed exists, when growing in fermentation liquor, for producing the recombinant microorganism organism of ω-hydroxy fatty acid derivative in vivo, described microbial organisms comprises the approach of at least 3 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises:
The acyl-acp reductase of (a) EC1.2.1.42;
The alcohol dehydrogenase of (b) EC1.1.-.-; And
Modified ω-the hydroxylase of (c) EC1.14.15.3.
40. recombinant microorganism organisms according to claim 39, wherein said modified ω-hydroxylase has the enzymatic activity of modified Cytochrome P450, and the ω-position of catalytic hydrocarbon chain effectively in vivo.
41. recombinant microorganism organisms according to claim 40, wherein said modified ω-hydroxylase is CYP153A-reductase hybrid fusion polypeptide.
42. recombinant microorganism organisms according to claim 41, wherein said CYP153A-reductase hybrid fusion polypeptide is self-centered CYP153A-RedRhF hybrid fusion protein matter.
43. recombinant microorganism organisms according to claim 39, wherein said modified ω-hydroxylase is the ω-hydroxylase hybrid fusion protein qualitative change body of EC1.14.15.3.
44. recombinant microorganism organisms according to claim 43, wherein said ω-hydroxylase hybrid fusion protein qualitative change body and SEQIDNO:6 have the sequence identity of at least 90%, and have the one or more sudden changes be selected from V141I, V141T, V141Q, V141G, V141M, V141L, R27L, R82D, R178N, A231T, N309R, N407A, V415R, T516V, P666A, P666D and A796V.
45. recombinant microorganism organisms according to claim 39, wherein said ω-hydroxy fatty acid derivative is α, omega-diol.
46. cell cultures comprising the microbial organisms described in any one of claim 39 to 45.
The method of 47. production ω-hydroxy fatty acid derivatives, it comprises:
A () provides recombinant microorganism organism in fermentation liquor, described microbial organisms comprises the approach of at least 3 kinds of nucleotide sequences of expressing following polypeptide of encoding through transformation, and described polypeptide comprises the acyl-acp reductase of EC1.2.1.42; The alcohol dehydrogenase of EC1.1.-.-; And the modified ω-hydroxylase of EC1.14.15.3;
B the reproducible feed comprising carbon source adds in described fermentation liquor by (); And
C () is by described separation of fermentative broth ω-hydroxy fatty acid derivative.
48. methods according to claim 47, wherein said ω-hydroxy fatty acid derivative is α, omega-diol.
49. methods according to any one of claim 47-48, wherein said reproducible feed is carbon back.
50. methods according to claim 49, the reproducible feed of wherein said carbon back is selected from corn, rattan, switchgrass, fresh stover, straw, timber, sewage, rubbish, cellulose municipal waste, waste gas, synthetic gas and carbon dioxide.
51. methods according to claim 47, wherein said carbon source is selected from glucose, fructose, mannose, galactose, wood sugar, arabinose, FOS, galactooligosaccharide, starch, cellulose, colloid, xylan, sucrose, maltose, cellobiose, turanose, hemicellulose, methylcellulose, sodium carboxymethyl cellulose, succinate, lactate, acetic acid esters, ethanol, methyl alcohol, glycerine and their potpourri.
52. methods according to claim 47, wherein said carbon source is glucose, glycerine or sucrose.
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